Chimeric and humanized anti-human CTLA4 monoclonal antibodies and uses thereof

ABSTRACT

This invention relates to compositions of chimeric and humanized antibodies that bind to the human CTLA4 molecule and their use in cancer immunotherapy and for reduction of autoimmune side effects compared to other immunotherapeutic agents.

FIELD OF THE INVENTION

This invention relates to chimeric and humanized antibodies that bind tothe human CTLA4 molecule and to methods of their use.

BACKGROUND OF THE INVENTION

The immune system of humans and other mammals is responsible forproviding protection against infection and disease. Such protection isprovided both by a humoral immune response and by a cell-mediated immuneresponse. The humoral response results in the production of antibodiesand other biomolecules that are capable of recognizing and neutralizingforeign targets (antigens). In contrast, the cell-mediated immuneresponse involves the activation of macrophages, neutrophil, naturalkiller cells (NK), and antigen-specific cytotoxic T-lymphocytes by Tcells, and the release of various cytokines in response to therecognition of an antigen.

The ability of T cells to optimally mediate an immune response againstan antigen requires two distinct signaling interactions. First, antigenthat has been arrayed on the surface of antigen-presenting cells (APC)must be presented to an antigen-specific naive T cells in the form ofMHC: peptide complex (1, 2). Such presentation delivers a signal via theT cell receptor (TCR) that directs the T cell to initiate an immuneresponse that will be specific to the presented antigen. Second, aseries of co-stimulatory signals, mediated through interactions betweenthe APC and distinct T cell surface molecules, triggers first theactivation and proliferation of the T cells and ultimately theirinhibition (3-5). Thus, the first signal confers specificity to theimmune response whereas the second signal serves to determine thenature, magnitude and duration of the response while limiting immunityto self. Of particular importance among these second signal molecules isbinding between the B7.1 (CD80) (6) and B7.2 (CD86) (7-9) ligands of theAntigen Presenting Cell and the CD28 and CTLA4 receptors (10-12) of theT-lymphocyte.

Cytotoxic T lymphocyte antigen-4 (CTLA4) is recognized as a keyregulators of adaptive immune responses, having a central role in themaintenance of peripheral tolerance and in shaping the repertoire ofemergent T cell responses and, therefore, a therapeutic target for thetreatment of cancer and inflammation. Treatment with anti-CTLA4antibodies has been shown to be a powerful tool for enhancing anti-tumorimmunity in preclinical models (10). Monotherapy with an antibodyagainst CTLA4 promoted rejection of transplantable tumors of variousorigins.

Based on promising preclinical tumor model studies, the clinicalpotential of antibodies against CTLA4 has been explored in differenthuman malignancies. Although anti-CTLA4 (Ipilimumab, marketed as Yervoy)has demonstrated efficacy in treating melanoma, treatment and targetingof CTLA4 is associated with autoimmune like toxicities. Characteristicside effects from inhibition of CTLA4 are generally calledimmune-related adverse events (irAEs) and the most common irAEs are skinrash, hepatitis, colitis and endocrinopathies, particularlyhypopituitarism. Therefore, there is a desire to improve the therapeuticpotential of anti-CTLA4 antibodies by increasing efficacy while reducingthe associated irAEs.

Another focus for the field of immunotherapy and the treatment oftumors, is the combination of different immune check inhibitors in orderto enhance anti-tumor activity, particularly against poorly immunogenictumors. However, this approach is associated with the risk of furtherincreasing the autoimmune side effects further highlighting the need toselectively modulate cancer immunity without enhancing autoimmunity.

Further investigations into the ligands of the CD28 receptor have led tothe identification and characterization of a set of related B7 molecules(the “B7 Superfamily”) (32-33). There are currently several knownmembers of the family: B7.1 (CD80), B7.2 (CD86), the inducibleco-stimulator ligand (ICOS-L), the programmed death-1 ligand (PD-L1;B7-H1), the programmed death-2 ligand (PD-L2; B7-DC), B7-H3, B7-H4 andB7-H6 (35-36).

B7-H1 is broadly expressed in different human and mouse tissues, such asheart, placenta, muscle, fetal liver, spleen, lymph nodes, and thymusfor both species as well as liver, lung, and kidney in mouse only (37).B7-H1 (PD-L1, CD274) is a particularly significant member of the B7Superfamily as it is pivotally involved in shaping the immune responseto tumors (38; U.S. Pat. Nos. 6,803,192; 7,794,710; United States PatentApplication Publication Nos. 2005/0059051; 2009/0055944; 2009/0274666;2009/0313687; PCT Publication No. WO 01/39722; WO 02/086083).

Programmed Death-1 (“PD-1”) is a receptor of B7-H1 as well as B7-DC.PD-1 is a type I membrane protein member of the extended CD28/CTLA4family of T cell regulators (39; United States Patent ApplicationPublication No. 2007/0202100; 2008/0311117; 2009/00110667; U.S. Pat.Nos. 6,808,710; 7,101,550; 7,488,802; 7,635,757; 7,722,868; PCTPublication No. WO 01/14557). Compared to CTLA4, PD-1 more broadlynegatively regulates immune responses. PD-1 is expressed on activated Tcells, B cells, and monocytes (40-41) and at low levels in naturalkiller (NK) T cells (42-43).

Interaction of B7-H1 and PD-1 has been found to provide a crucialnegative co-stimulatory signal to T and B cells (43) and functions as acell death inducer (39). The role of B7-H1 and PD-1 in inhibiting T cellactivation and proliferation has suggested that these biomolecules mightserve as therapeutic targets for treatments of inflammation and cancer.Consequently, the use of anti-PD1 and anti-B7-H1 antibodies to treatinfections and tumors and up-modulate an adaptive immune response hasbeen proposed and demonstrated to be effective for the treatment of anumber of human tumors. However, not all subjects respond or havecomplete responses to anti-PD-1 or anti-B7-H1 treatment and so there isa strong interest in combining anti-PD-1 or anti-B7-H1 antibodies withother immune check inhibitors in order to enhance anti-tumor activity.

4-1BB (also known as CD137 and TNFRSF9) is another immune checkpointmolecule. The best characterized activity of CD137 is its costimulatoryactivity for activated T cells. Crosslinking of CD137 enhances T cellproliferation, IL-2 secretion, survival and cytolytic activity. Further,like anti-CTLA4, anti-4-1BB antibodies can enhance immune activity toeliminate tumors in mice (27-29). However, unlike the tendency ofanti-CTLA4 antibodies to exacerbate autoimmune diseases, cancertherapeutic anti-4-1BB mAbs have been shown to abrogate the developmentof autoimmune diseases in lupus prone mice, in which they inhibitedanti-dsDNA antibody production and reduced the renal pathology (25, 26).Previously data have demonstrated that it is possible to reduce theautoimmune side effects of anti-CTLA4 treatment in a mouse colon cancertumor model by combining treatment of anti-CTLA4 with anti-4-1BBantibody, while enhancing the anti-tumor activity (19). Thisdemonstrates that it is possible to offset the autoimmune side effectsof anti-CTLA4 tumor therapy.

Preclinical screening of anti-human CTLA4 antibodies is fraught withdifficulty because in vitro immunological correlates are sometimes oflittle value, as demonstrated by experience with anti-mouse CTLA4antibodies. The same anti-mouse CTLA4 antibodies that induce potentanti-tumor immunity in vivo can have variable effects on T cells invitro. Anti-CTLA4 antibodies enhanced T cell proliferation in responseto alloantigen, but suppressed T cell proliferation in response tocostimulation by anti-CD 28 (30, 31). Also, CTLA4 engagement withantibody could either promote or inhibit proliferation of differentsubsets of T cells in the same culture (32). This complication can beovercome if one can study human T cell responses in a rodent model.

Described herein are anti-CTLA4 antibodies with reduced autoimmune sideeffects when used to enhance immune responses and for use in anti-tumortherapy. Furthermore, these antibodies can be used in combination withother checkpoint inhibitors, such as anti-PD-1 and anti-4-1BB, toenhance anti-tumor while abrogating autoimmune side effects.

SUMMARY OF THE INVENTION

This invention relates to antibody compositions and theirantigen-binding fragments that bind to the human CTLA4 molecule andtheir use for cancer immunotherapy with reduced autoimmune side effects.Specifically, the invention relates to antibodies with enhanced CTLA4blocking activity for CTLA4 ligands B7.1 and B7.2, enhanced effectorfunction, or reduced binding to soluble CTLA4 relative to membrane boundor immobilized CTLA4.

The antibody may comprise a light chain variable amino acid sequencehaving the amino acid sequence comprising a light chain variable aminoacid sequence having the amino acid sequence set forth in SEQ ID NO: 1,and a heavy chain variable amino acid sequence having the amino acidsequence set forth in SEQ ID NO: 2. The antibody may also comprise aheavy chain variable amino acid sequence having the amino acid sequenceset forth in SEQ ID NO: 27, 28 or 29, and a light chain variable aminoacid sequence having the amino acid sequence set forth in SEQ ID NO: 30,31 or 32. The antibody may comprise a light chain variable region havingCDR sequences set forth in SEQ ID NOS: 21, 22 and 23, and a heavy chainvariable region having CDR sequences set forth in SEQ ID NOS: 24, 25 and26. More specifically, the antibody may comprise a heavy chain variableregion having a CDR2 sequence set forth in SEQ ID NO: 33, 34 or 35, anda light chain variable region having CDR sequences set forth in SEQ IDNO: 36, 37 or 38.

The immunoglobulin heavy chain constant regions of the antibody maycomprise the amino acid sequence set forth in SEQ ID NO: 3 or 4. Theimmunoglobulin heavy chain constant region of the antibody may alsocomprise a mutation. Relative to the sequence of the hIgG1 backbone inSEQ ID NO: 3, the mutation may be M135Y, S137T, T139E, S181A, E216A, orK217A, or a combination thereof. Preferably, the immunoglobulin heavychain constant region of the antibody may comprise all six mutations.The antibody may comprise a heavy chain amino acid sequence having theamino acid sequence set forth in SEQ ID NO: 6, and a light chain aminoacid sequence having the amino acid sequence set forth in SEQ ID NO: 8.The antibody may also comprise a heavy chain amino acid sequence havingthe amino acid sequence set forth in SEQ ID NO: 9, 11 or 13, and a lightchain amino acid sequence having the amino acid sequence set forth inSEQ ID NO: 15, 17 or 19. The antibody may be capable of binding humanCTLA4. The antibody may also inhibit binding of human CTLA4 to B7-1 orB7-2.

Further provided herein is an antigen binding fragment of the antibodiesdescribed herein.

Also provided herein is a pharmaceutical composition comprising atherapeutically effective amount of the antibodies described herein. Thepharmaceutical composition may comprise a physiologically acceptablecarrier or excipient.

In another aspect, presented herein are methods for enhancing one ormore immune functions or responses in a subject, comprisingadministering to a subject in need thereof the anti-CTLA4 antibodycompositions and pharmaceutical compositions described herein. In aspecific embodiment, presented herein are methods for preventing,treating, and/or managing a disease in which it is desirable to activateor enhance one or more immune functions or responses. The disease may bea cancer, which may be a human malignancy. In particular, the humanmalignancy may be melanoma, lung cancer, breast cancer, hepatocellularcarcinoma, ovarian carcinoma, prostate carcinoma, Hodgkin's ornon-Hodgkin's lymphoma, acute myelogenic leukemia, chronic myelogenicleukemia, acute lymphocytic leukemia, chronic lymphocytic leukemia, orrenal cell carcinoma. In another embodiment, the disease to be treatedis an infectious disease. The method described herein may minimizeautoimmune adverse effects associated with immunotherapy.

In other specific embodiments, the method comprises combination therapy,wherein the anti-CTLA4 antibody compositions described herein areadministered to a subject in combination with another therapy, which mayactivate or enhance one or more immune functions or responses. Inanother embodiment, the anti-CTLA4 antibody compositions describedherein are administered as an adjuvant in combination with an antigeniccomposition. In a particular embodiment, the anti-CTLA4 antibodycompositions described herein are administered in combination with avaccine composition to induce or activate or enhance the immune responseelicited by the vaccine composition.

In a specific embodiment, the anti-CTLA4 antibody compositions describedherein are administered to a subject in combination with one or moreother therapies that target different immunomodulatory pathways. In apreferred embodiment, the activity of the therapy targeting a differentimmunomodulatory pathway is complementary or synergistic with theanti-CTLA4 antibody compositions described herein. In one instance, theanti-CTLA4 antibody compositions described herein are administered incombination with other checkpoint inhibitors or small oncoimmunologicalmodulators such as indoleamine 2,3-dioxygenase (IDO) inhibitors. Inanother instance, the anti-CTLA4 antibody compositions described hereinare administered in combination with immune stimulating molecules.Specific embodiments include combining the anti-CTLA4 antibodycompositions described herein with anti-PD-1 (pembrolizumab (Keytruda)or Nivolumab (Opdivo)), anti-B7-H1 (atezolizumab (Tecentriq) ordurvalumab), anti-B7-H3, anti-B7-H4, anti-LAG3, anti-Tim3, anti-CD40,anti-OX40, anti-BTLA, anti-CD27, anti-ICOS or anti-41BB. In anotherembodiment, the anti-CTLA4 antibody compositions described herein andthe second immune stimulating molecule are combined in a singlebi-specific antibody.

In another embodiment, an anti-human CTLA4 antibody described herein maypreferentially bind to human CTLA-4 expressed on the cell surfacerelative to soluble CTLA4 molecules. The anti-human CTLA4 antibody maybind to human CTLA4 and preferentially upregulate the expression of B7.1or B7.2 in vivo. The antibody may be contained in a composition for usein modulating immune responses (immunotherapy) and the treatment ofcancer.

The invention further concerns the method of screening for anti-humanCTLA4 mAbs with preferred activity. Preclinical screening for anti-humanCTLA4 mAbs is fraught with difficulty because in vitro immunologicalcorrelates for cancer immunity and autoimmune adverse effect are notdefined. Significant autoimmune side-effects have been observed inclinical trials with human anti-CTLA4 (Ipilimumab), especially whencombined with anti-PD-1. In order to identify anti-CTLA4 antibodies withreduced immune related toxicities, antibodies demonstrating anti-tumoractivity in humanized mice can be screened for their ability to reduceautoimmune adverse effects in vivo using human CTLA4 gene knock-in mice.

In another embodiment, the invention concerns a method of screening foranti-human CTLA4 mAbs with enhanced anti-tumor effect wherein theantibodies demonstrate enhanced local depletion of Treg cells in thetumor environment.

In yet another embodiment, the invention concerns methods of monitoringthe blocking effects of anti-CTLA4 antibodies in vivo by monitoring theexpression levels of B7.1 and B7.2 on immune cells such as antigenpresenting cells (APCs). The invention further contemplates biomarkersfor measuring the biological activity of anti-CTLA4 antibodies in vivoand monitoring patent responses to anti-CTLA4 treatment by measuring thelevel B7.1 and B7.2 expression on immune cells ex vivo.

In order to map the CTLA4 binding epitope of the L3D10 parent antibodyand the humanized variants, PP4631 and PP4637, the fact that the mouseand human CTLA4 proteins are cross-reactive to B7-1, but not to theanti-CTLA-4 antibodies was exploited. Accordingly, a number of mutantsof the human CTLA-4Fc protein were designed in which clusters of aminoacids from the human CTLA-4 protein were replaced with amino acids fromthe murine Ctla-4 protein. As the anti-CTLA-4 antibodies used in thisstudy do not bind to murine Ctla-4, binding of the anti-human CTLA-4antibodies can be abolished when key residues of the antibody bindingepitope are replaced with murine amino acids.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 . Schematic diagram of the chimeric (left) and humanized (right)L3D10 antibodies with a novel combination of mutations in the IgG1 Fcregion. The positions of the mutations in the Fc region are identifiedby their amino acid position number and the amino acids are identifiedby their single letter code, with the letter before the numberrepresenting the replace amino acid and the letter after the numberrepresenting the introduced amino acid. The variable region of theantibodies is depicted with open ovals and the human sequence isdepicted with gray rectangles. V=variable region; C=constant region;L=light chain; H=heavy chain.

FIG. 2 . CTLA4 Binding of chimeric L3D10 and 10D1 to plate immobilizedCTLA4, as determined by ELISA. ELISA plates were coated with 1 μg/ml ofCTLA4-His protein (Sino Biological, China). The given concentration ofbiotinylated binding proteins were added and binding was measured usingHRP-conjugated streptavidin. 10D1-1 and -2 are two independent materiallots of the ‘same antibody. B7.1-Fc is a positive control and Fc is anegative control.

FIG. 3 . L3D10 competition assay. 10D1 is less efficient in blockingchimeric L3D10 binding to CTLA4 than chimeric L3D10. The experiment wasperformed as in FIG. 2 , except that biotinylated chimeric L3D10 wasmixed with the given concentration of unlabeled CTLA4-binding proteinsor CTLA4-Fc prior to adding to the ELISA plates. Note much betterblocking by unlabeled L3D10 than 10D1, which suggest that these antibodybinding sites are not identical.

FIG. 4 . Blocking CTLA4 binding to plate immobilized B7.1. B7.1 Fcprotein was coated onto ELISA plates at 0.5 μg/ml. After washing andblocking, biotinylated CTLA4-Fc protein was added at 0.25 μg/ml in thepresence of given concentrations of the competing proteins. Data shownare means of duplicate optical density at 405 nM. Whereas B7.1-Fc,chimeric L3D10 and CTLA4-Fc all block the CTLA4:B7.1 interaction in adose-dependent manner, two separate lots of 10D1 antibody failed toblock at all doses tested. Biotinylation of CTLA4 does not destroy 10D1epitopes on CTLA4 as both lots of 10D1 show strong binding tobiotinylated CTLA4 (data not shown).

FIG. 5 . Blocking CTLA4 binding to plate immobilized B7.2. B7.2Fcprotein was coated onto ELISA plates at 0.5 μg/ml. After washing andblocking, biotinylated CTLA4-Fc protein was added at 0.25 μg/ml in thepresence of given concentrations of the competing proteins. Whereaschimeric L3D10 blocks the CTLA4:B7.2 interaction in a dose-dependentmanner, two separate lots of 10D1 antibody failed to completely blockthe CTLA4:B7.2 interaction even at the highest concentration.

FIG. 6 . Both 10D1 and L3D10 potently block B7-CTLA4 interaction usingsoluble B7-1 and B7-2 and immobilized CTLA4-Fc. Varying doses ofanti-human CTLA4 mAbs were added along with 0.25 μg/ml of biotinylatedhuman CTLA4-Fc to plate-coated with human B7-1Fc. The amounts of CTLA4bound to plates were measured using HRP-conjugated streptavidin. Datashown are means of duplicates and are representative of two independentexperiments.

FIG. 7 . Blocking CTLA4 binding to cell surface expressed B7.1.Biotinylated CTLA4-Fc protein was added to B7.1 expressing CHO cells at0.5 μg/ml in the presence of the given concentration of the competingproteins. Binding of biotinylated fusion protein to CHO cellstransfected with mouse or human B7-1 and B7-2 was detected by flowcytometry. The amounts of bound receptors were measured usingphycoethrythorin-conjugated streptavidin. Data shown are meansfluorescence intensity of triplicate samples. Whereas chimeric L3D10blocks the CTLA4:B7.1 interaction in a dose-dependent manner, twoseparate lots of 10D1 antibody failed to block at all doses tested.

FIG. 8 . Blocking CTLA4 binding to cell surface expressed murine B7.1.Modest but detectable blocking of mouse B7-1-human CTLA4 interaction by10D1 when mB7-1 is expressed on CHO cells. Varying doses of anti-humanCTLA4 mAbs were added along with 0.25 μg/ml of human CTLA4-Fc to CHOcells expressing mouse B7-1. Data shown are means and SEM or triplicatedata and are representative of two independent experiments.

FIG. 9 . Blocking CTLA4 binding to cell surface expressed B7.2.Biotinylated CTLA4-Fc protein was added to B7.2 expressing CHO cells at0.5 μg/ml in the presence of the given concentration of the competingproteins. Whereas chimeric L3D10 blocks the CTLA4:B7.2 interaction in adose-dependent manner, two separate lots of 10D1 antibody failed tocompletely block the CTLA4:B7.2 interaction even at the highestconcentration. Data shown in this figure has been repeated at least 5times.

FIG. 10 . 10D1 binds to biotinylated human CTLA4-Fc better than L3D10.Varying doses of anti-human CTLA4 mAbs or control IgG were coated ontothe plate. Biotinylated CTLA4-Fc was added at 0.25 μg/ml. The amounts ofCTLA4 bound to plates were measured using HRP-conjugated streptavidin.Data shown are means of duplicates and are representative of twoindependent experiments.

FIG. 11 . L3D10 but not 10D1 blocks the interaction betweenpolyhistindine tagged CTLA4 and CHO cells expressing human B7-1. CHOcells expressing human B7-1 were incubated with polyhistidine-taggedCTLA4 along with given doses of antibodies, the amounts of CTLA4-Fc weredetected with PE-streptavidin and measured by FACSCanto II. Data shownare means fluorescence intensity of triplicate samples and arerepresentative of two independent experiments.

FIG. 12 . Chimeric L3D10 induces complete remission of establishedtumors in the syngeneic MC38 model. Top panel depicts experimentaldesign and the lower panels show growth kinetics of MC38 tumors in micethat received either control IgG (lower left panel, n=6) or chimericL3D10 (lower right panel, n=5).

FIG. 13 . Therapeutic effect of chimeric L3D10 and 10D1 in the MC38tumor model. Human CTLA4-knock-in mice with body weight of approximately20 grams were used for the study. 1×10⁶ MC38 tumor cells were injectedsubcutaneously into Ctla4^(h/h) mice and when the tumor reached a sizeof 0.5 cm in diameter, tumor bearing mice were randomized into threegroups with 5 or 6 mice each. Mice were then treated (i.p.) with 100μg/injection of 10D1, chimeric L3D10 or control hIgGFc on days 7, 10,13, and 16 as indicated by the arrows. The results of duplicate exptsare shown (left and right panels) and data shown are means and S.D. oftumor size (n=6 per group in the left panel, n=5 per group in the rightpanel). L3D10 and 10D1 have similar therapeutic effect in this model andare both able to induce complete remission of established tumors. Thediameters (d) of the tumor were calculated using the following formula:D=J (ab), V=ab2/2, where a is the long diameter, while b is the shortdiameter. Statistical analyses were performed by two-way repeatedmeasures ANOVA (treatment×time). For the left panel: P=10D1 vs. hIgGFc:5.71e−07; L3D10 vs. hIgGFc: P=5.53e−07; 10D1 vs. L3D10: P=0.869.

FIG. 14 . Effective rejection of MC38 by anti-CTLA-4 mAbs in CTLA4^(h/m)mice. As in FIG. 13 , except that heterozygous CTLA4^(h/m) mice areused. Data shown are means and SEM of tumor diameters (6 mice pergroup); 10D1 vs. hIgGFc: P=0.0011; L3D10 vs. hIgGFc: P=5.55e−05; 10D1vs. L3D10: P=0.0346.

FIG. 15 . Therapeutic effect of chimeric L3D10 and 10D1 in the B16-F1melanoma tumor model. Human CTLA4-knockin mice with body weight ofapproximately 20 grams were used for the study. Arrows indicate the timeof treatment (50 μg/mice/treatment). Data shown are means and S.D. ofthe tumor size (n=4 per group). L3D10 have similar therapeutic effect inthis model and are both able to delay tumor growth in this aggressiveand poorly immunogenic tumor model.

FIG. 16 . Assay for measuring CTLA4 blocking in vivo. B7.1 or B7.2 bindson dendritic cells bind to, and are down-regulated by, CTLA4 on surfaceof T cells. However, binding of blocking anti-CTLA4 antibodies preventsB7.1/B7.2 binding to CTLA4 and thus prevents the downregulation of B7.1and B7.2, resulting in a net increase in B7.1/B7.2 expression. However,with chimeric T cells expressing both human and mouse CTLA4, antibodiesthat bind human CTLA4 do not prevent B7.1/B7.2 binding to the murineCTLA4, which restores B7.1/B7.2 inhibition.

FIGS. 17A-F. 10D1 does not block B7-CTLA4 interaction in vivo. Using theassay described in FIG. 11 , cells from mice treated with anti-CTLA4antibodies were used to assay B7.1 and B7.2 expression. FIG. 17A shows adiagram of experimental design. Briefly, age and gender-matched micereceived 500 μg of antibodies or their controls intraperitoneally. At 24hours after injection, mice were sacrificed and their spleen cells werestained with anti-CD11c, CD11b, anti-B7-1 and anti-B7-2 mAbs. FIG. 17Bshows representative data showing the phenotype of CD11c^(hi) DCanalyzed for B7 expression.

FIG. 17C shows representative histograms depicting the levels of B7-1 onDC from mice that received control IgG1-Fc, L3D10 or 10D1. Data in thetop panel shown antibody effect in homozygous knockin mice, while thatin the bottom panel show antibody effect in the heterozygous mice. FIG.17D shows as in FIG. 17C, except that expression of B7-2 is shown. Datashown in FIGS. 17C and D are representative of those from 3 mice pergroup and have been repeated once with three mice per group.

FIG. 17E shows that in human CTLA4 homozygous mice, L3D10 but not 10D1induced expression of B7-1 (left panel) and B7-2 (right panel). Datashown are summarized from two experiments involving a total of 6 miceper group. In each experiment, the mean data in the control mice isartificially defined as 100% and those in experimental groups arenormalized against the control. FIG. 17F as in FIG. 17E, except thatheterozygous mice are used. Neither L3D10 nor 10D1 block B7-CTLA4interaction in mice that co-dominantly express both mouse and humanCtla4 genes.

FIG. 18 . L3D10 binds to human but not mouse CTLA4. Data showing are dotplots of intracellular staining of CTLA4 among gated Cd3⁺Cd4⁺ cells,using spleen cells from Ctla4^(h/h) (top) or Ctla4^(m/m) (bottom) mice.Anti-mouse CTLA4 mAb 4F10 was used as control.

FIG. 19 . Therapeutic effect of chimeric L3D10 and 10D1 in CTLA4^(h/m)mice. The top panel depicts the experimental design. The Ctla4h/h micewere challenged with colon cancer cell line MC38 and when the tumorreached a size of approximately 5 mm in diameter, the mice were treated4 times with control human IgG-Fc, L3D10 or 10D1 and observed tumor sizeover a 6 weeks period. The lower panels shows the growth kinetics ofMC38 tumors in mice that received either control IgG, chimeric L3D10 or10D1 (n=6 per group). Despite apparent differences in CTLA4 blockingactivity in vivo as shown in FIG. 16 , both L3D10 and 10D1 displaystrong anti-tumor activity against the MC38 model in chimericCTLA4^(m/h) mice.

FIGS. 20A-B. 10D1 and L3D10 have similar therapeutic effect on B16melanoma growth. 1×10⁵ B16 tumor cells were injected (s.c.) intoCtla4^(h/h) mice (n=4-5), and treated (i.p.) with 100 μg (FIG. 20A) or250 μg (FIG. 20B) 10D1, L3D10 or control IgGFc on day 11,14,17 (FIG.20A) or on day 2, 5, and 8 (FIG. 20B), as indicated by arrows. For FIG.20A, 10D1 vs. hIgGFc: P=0.0265; L3D10 vs. hIgGFc: 10D1 vs. L3D10:P=0.0487; P=0.302. For FIG. 20B, 10D1 vs. hIgGFc: P=0.00616; L3D10 vs.hIgGFc: P=0.0269: 10D1 vs. L3D10: P=0.370. Data represent mean±SEM of4-5 mice per group. Statistical analyses were performed by two-wayrepeated measures ANOVA.

FIGS. 21A-B. Immunotherapeutic effects between L3D10 and 10D1 inCtla4^(h/h) (FIG. 21A) and Ctla4^(m/h) (FIG. 21B) in mice that wereterminated before rejection in complete in order to evaluate depletionof Treg within tumor microenvironment. Data shown are means and SEM oftumor diameters of two independent experiments, involving 5 mice pergroup.

FIGS. 22A-F. Blocking the B7-CTLA4 interaction does not contribute tocancer immunotherapeutic activity of anti-CTLA4 mAb. FIG. 22A showscomparable immunotherapeutic effect despite vastly different blockingactivity by two anti-CTLA4 mAbs. 5×10⁵ MC38 tumor cells were injected(s.c.) into Ctla4^(h/h) mice (n=6), and treated (i.p.) with 100 μg 10D1,L3D10 or control hIgG-Fc on days 7, 10, 13, and 16, as indicated byarrows. Data represent mean±SEM of six mice per group. Statisticalanalyses were performed by two-way repeated measures ANOVA(treatment×time). 10D1 vs. hIgG-Fc: P=5.71e−07; L3D10 vs. hIgG-Fc:P=5.53e−⁰⁷; 10D1 vs. L3D10: P=0.869. Data are representative of threeindependent experiments. FIG. 22B. In mice that neither antibodies blockB7-CTLA4 interaction, both induce robust tumor rejection. As in FIG.22A, except that heterozygous mice that express both mouse and humanCTLA4 were used. 10D1 vs. hIgG-Fc: P=0.0011; L3D10 vs. hIgG-Fc:P=5.55e−⁰⁵; 10D1 vs. L3D10: P=0.0346. Data are representative of threeindependent experiments.

FIGS. 22C-F, Blocking B7-CTLA4 interaction does not contribute toselective depletion of Treg in tumor microenvironment. FIGS. 22C and D.Regardless of their ability to block B7-CTLA4 interaction, L3D10 and10D1 do not delete Treg in the spleen. Data shown are % of Foxp3+ cellsamong spleen CD4 T cells in Ctla4^(h/h) (FIG. 22C) and Ctla4^(m/h) (FIG.22D) mice. n=6. e and f, both L3D10 and 10D1 delete Treg among tumorinfiltrating CD4 T cells in Ctla4^(h/h) (FIG. 22E) and Ctla4^(m/h) (FIG.22F) mice. Data shown in c-f are % of Treg at 17 (experiment 1) or 19days (experiment 2) after tumor cell challenge and 10 or 12 days afterinitiation of 4 anti-CTLA4 mAb treatments as indicated in arrows.

FIGS. 23A-F. Evaluation of blocking activities of commonly usedanti-mouse CTLA4 mAbs 9H10 and 9D9. FIGS. 23A and B show that 9H10 doesnot block B7-CTLA4 interaction if B7-1 (FIG. 23A) and B7-2 (FIG. 23B)are coated onto plates. Biotinylated mouse CTLA4-Fc fusion protein wereincubated with B7-coated plates in the presence of given concentrationof control IgG or anti-mouse CTLA4 mAb 9D9 and 9H10. The CTLA4 bindingis detected with HRP-conjugated streptavidin. Data shown are means ofduplicated and are representative of two independent experiments. FIGS.23C and D show that 9D9 and 9H10 exhibit differential binding to soluble(FIG. 23C) and plate bound CTLA4-Fc (FIG. 23D). Data shown are means ofduplicated and are representative of at least two independentexperiments. FIGS. 23E and F show the effects of anti-mouse CTLA4 mAbs9D9 and 9H10 on levels of B7-1 (FIG. 23E) and B7-2 (FIG. 23F) onCD11c^(hi) DC from WT (Ctla4^(m)/m) spleen cells at 24 hours aftertreatment with 500 μg of antibodies i.p. The data are summarized from 6independent mice per group in two independent experiments involving 3mice per group each.

FIGS. 24A-D. Distinct in vivo and in vivo blocking activities ofanti-mouse CTLA4 mAb 4F10. FIGS. 24A and B show the effect of 4F10 oninteraction of CTLA4-Fc to plate-coated B7-1 (FIG. 24A) or B7-2 (FIG.24B). Biotinylated mouse CTLA4-Fc fusion protein were incubated withB7-coated plates in the presence of given concentration of control IgGor anti-mouse CTLA4 mAb 4F10. The CTLA4 binding is detected withHRP-conjugated streptavidin. Data shown are means of duplicated and arerepresentative of two independent experiments. FIGS. 24C and D show theimpact of 4F10 on B7-1 and B7-2 expression. Summary data on B7-1 (FIG.24C) and B7-2 (FIG. 24D) levels from 6 mice per group. The B7 levels inthe control IgG-treated group are artificially defined as 100%.

FIG. 25 . Adverse effects of chimeric L3D10 and 10D1 in combination withanti-PD-1. Top panel depicts the experimental design. 10-day oldfemale-only human CTLA4-knockin mice with body weight of greater than 4grams were used for the study. They received indicated proteins or theircombinations. Arrows indicate time of treatment (100 μg/mice/treatment).Data shown are means and S.D. of % weight gains. Chimeric L3D10 and 10D1have comparable cancer therapeutic effect in adult mice (FIG. 13 ) butdistinct adverse effects are seen when 10D1 is combined with theanti-PD-1 mAb.

FIG. 26 . Adverse effects of chimeric L3D10 and 10D1 in combination withanti-PD-1. The graph shows the terminal body weight on Day 42 in themice from the experiment outlined in FIG. 25 that received eithercontrol IgG, 10D1+anti-PD-1 or chimeric L3D10+anti-PD-1 (n=5 per group).A significant reduction in weight is observed with the anti-PD1+10D1combination, which was not seen with the anti-PD-1+Chimeric L3D10combination.

FIG. 27 . Pathological effects of chimeric L3D10 and 10D1 in combinationwith anti-PD-1. To further examine to relative toxicity of L3D10compared to 10D1 when administered in combination with anti-PD-1, welooked at the gross anatomy of the mice described in FIG. 26 above. TheUterus/Ovary/Bladder and thymus were noticably smaller in mice treatedwith 10D1+PD-1, whereas the organs in mice treated with L3D10+anti-PD-1was comparable to hIgG control. In contrast, the hearts dissected frommice treated with 10D1 appeared larger in size with a noticeably whiterappearance.

FIGS. 28A-D. Treatment with 10D1 in combination with anti-PD-1 resultsin abnormal erythropoiesis. Given the differences in the hearts observedin FIG. 27 , we looked at erythropoiesis within the mice and observedclear differences in the mice treated with 10D1+anti-PD-1 relative tothe groups treated with L3D10+anti-PD-1 or control antibody (hIgG),which were fairly similar. The bone marrow from mice treated with10D1+anti-PD-1 had a noticeably whiter color (FIG. 28A) and the isolatedblood was almost completely white in color (FIG. 28B). In accordancewith this, when we analyzed differentiation of the red blood cells usingdistribution of CD119 and CD71 markers we observed a statisticallysignificant reduction in the number of cells undergoing Stage IVdevelopment in the 10D1+anti-PD-1 treated mice. Representative FACSprofiles are shown in FIG. 28C, while summary data are presented in FIG.28D.

FIG. 29 . Flow cytometry analysis of anti-red blood cell antibodies.Blood samples from NOD.SCID.Il2rg−/− (NSG) mice were stained with plasmasamples from the mice that received antibody treatment during theperinatal period. Sera from NSG mice and those without sera were used asnegative control. All sera were used at 1:50 dilution. These data showthat no mice produced anti-red cell antibodies.

FIG. 30 . Pathology of the heart in mice treated with chimeric L3D10 and10D1 in combination with anti-PD-1. To further determine the toxicologyof L3D10 vs 10D1 in combination with anti-PD-1, we performedhistological analysis of the heart in mice described in FIG. 26 . Micetreated with 10D1+anti-PD-1 displayed a high level of T cellinfiltration that was not observed in mice treated with L3D10+anti-PD-1or mice treated with human IgG control.

FIG. 31 . Pathology of the lung in mice treated with chimeric L3D10 and10D1 in combination with anti-PD-1. To further determine the toxicologyof L3D10 vs 10D1 in combination with anti-PD-1, we performedhistological analysis of the lung in mice described in FIG. 26 . Micetreated with 10D1+anti-PD-1 displayed a high level of T cellinfiltration that was not observed in mice treated with L3D10+anti-PD-1or mice treated with human IgG control.

FIG. 32 . Pathology of the salivary gland in mice treated with chimericL3D10 and 10D1 in combination with anti-PD-1. To further determine thetoxicology of L3D10 vs 10D1 in combination with anti-PD-1, we performedhistological analysis of the salivary in mice described in FIG. 26 .Mice treated with 10D1+anti-PD-1 displayed a much higher level of T cellinfiltration than observed in mice treated with L3D10+anti-PD-1 or micetreated with human IgG control.

FIGS. 33A-F. Pathology of the kidney and liver in mice treated withchimeric L3D10 and 10D1 in combination with anti-PD-1. To furtherdetermine the toxicology of L3D10 vs 10D1 in combination with anti-PD-1,we performed histological analysis of the kidney and liver in micedescribed in FIG. 26 . FIGS. 33A-C are sections from the kidney andFIGS. 33D-E are sections taken from the liver. Mice treated with10D1+anti-PD-1 displayed a high level of T cell infiltration thanobserved in mice treated with L3D10+anti-PD-1 or mice treated with humanIgG control.

FIG. 34 . Toxicity scores of mice treated with chimeric L3D10 and 10D1in combination with anti-PD-1. This tissue data shown if FIGS. 30-33 issummarized and shows the high toxicity scores of mice treated with10D1+anti-PD-1 relative to L3D10+anti-PD-1 which has scores onlymarginally higher than the hIgG control mouse group.

FIG. 35 . 10D1+anti-PD-1 do not have significant toxicity in theCtla4^(h/m) mice as evidenced by normal body weight gains in mice thatreceived antibody treatment during the perinatal period. The micereceived treatments with given antibody or combinations on days 10, 13,16, 19 and 22 intraperitoneally (100 μg/mice/injection/antibody). Micewere weighed at least once every 3 days.

FIG. 36 . L3D10 and 10D1 display similar binding patterns for plateimmobilized CTLA4. ELISA plates were coated with 1 μg/ml of CTLA4-Hisprotein (Sino Biological, China). The given concentration ofbiotinylated binding proteins were added and binding measured usingHRP-conjugated streptavidin. 10D1-1 and -2 are two independent materiallots of the same antibody. hIgG-Fc is a human Ig negative control.

FIG. 37 . L3D10 displays reduced binding soluble CTLA4. Givenconcentration of anti-human CTLA4 mAbs were coated on the plateovernight, after washing and blocking with bovine serum albumin,biotinylated CTLA4-Fc was added at 0.25 μg/ml. After incubation answashing, the amounts of captured CTLA4-Fc were measured usingHRP-labeled streptavidin.

FIGS. 38A-B. Alignment of the humanized antibody variable regions withthe parental L3D10 antibody sequence. The heavy chain variable region(FIG. 38A)(SEQ ID NOS: 62-64) and light chain variable region (FIG.38B)(SEQ ID NOS: 70-72) of the humanized antibody sequences arealignment with the parental L3D10 antibody (heavy chain: SEQ ID NO: 57;light chain: SEQ ID NO: 65) and the respective human antibody frameworks(heavy chain: SEQ ID NOS: 58-61; light chain: SEQ ID NOS: 66-69). Aminoacid numbering is shown above the parental sequence. The three CDRs areshown in italics and underlined with dashes. Back mutations to the mouseparental sequence are in bold and underlined. Novel amino acids i.e.amino acid residues not present in the parental antibody sequence or therespective human antibody framework are shown in bold. Mutationsintroduced into the CDR2 sequences are shown in double underlined. CDRsequences are shown in red based on www.bioinf.org.uk/abs/.

FIGS. 39A-B. Anti-tumor activity of humanized L3D10 antibodies comparedto 10D1. Using the MC38 mouse tumor model in human CTLA4 knockin mice welooked at the anti-tumor activity of humanized L3D10 antibodies comparedto the chimeric L3D10 antibody and 10D1. The top panel shows thetreatment schedule of the in vivo experiment; mice were given a total of4 doses of antibody every 3 days starting on day 7 after inoculation.All humanized antibodies (n=6 per group) completely eradicated thetumors and were comparable to 10D1 (bottom panel).

FIG. 40 . Anti-tumor activity of humanized L3D10 antibodies inCTLA4^(h/m) mice. The top panel shows the treatment schedule of the invivo experiment; Ctla4^(h/m) mice received control hIg or one of threedifferent anti-human CTLA4 mabs at doses of 30 (−30, solid lines) or 10(−10, dotted lines) mg per injection at the indicated dates after MC38tumor injection. Tumor sizes were measured once every three days.

FIG. 41 . Therapeutic effect of anti-CTLA-4 mAb in minimal diseaseB16-F1 tumor model. Using the B16-F1 mouse tumor model in human CTLA4knockin mice we looked at the anti-tumor activity of humanized L3D10antibodies. 1×10⁵ B16 tumor cells were injected (s.c.) into Ctla4^(h/h)mice (n=5-6). On days 2, 5, and 8, the mice were treated with controlIg, 10D1, chimeric L3D10 or PP4637 and PP4638 (250 μg/mouse, i.p.).Tumor incidence and sizes were measured every other day. 10D1 vs.hIgGFc: P=0.00616; L3D10 vs. hIgGFc: P=0.0269; 10D1 vs. L3D10: P=0.370;PP4637 vs. hIgGFc: P=0.0005; PP4637 vs. 10D1: P=0.805; PP4638 vs.hIgGFc: P=0.0016; PP4638 vs. 10D1: P=0.856. Data represent mean±SEM of5-6 mice per group. Sizes of tumors were considered as 0 for mice thatnever developed tumor.

FIG. 42 . Comparison among 10D1, PP4631 and PP4637 females for theircombined toxicity with anti-PD-1 mAb. Female CTLA4^(h/h) mice weretreated on days 10 or 11 days after birth with 4 injections ofantibodies (100 μg/mice/injection, once every three days) or control Fcas specified in the legends. Mice were weighted once every 3 days. Datashown are means and SEM of % weight gain over a 30 day period. All micewere sacrificed on day 43 for histological analysis. The number of miceused per group is shown in the parentheses of labels.

The P values when the different treatment groups are compared are shownbelow:

hlg VS αPD1 + L3D10 P value = 0.16 hlg VS αPD1 + hlg P value = 0.0384*hlg VS αPD1 + 10D1 P value = <2e−16*** hlg VS αPD1 + PP4631 P value =0.16 hlg VS αPD1 + PP4637 P value = 0.00207** αPD1 + L3D10 VS αPD1 + hlgP value = 0.00654** αPD1 + L3D10 VS αPD1 + 10D1 P value = <2e−16***αPD1 + L3D10 VS αPD1 + PP4631 P value = 0.492 αPD1 + L3D10 VS αPD1 +PP4637 P value = 0.000124*** αPD1 + hlg VS αPD1 + 10D1 P value =<2e−16*** αPD1 + hlg VS αPD1 + PP4631 P value = 0.0579 αPD1 + hlg VSαPD1 + PP4637 P value = 0.409 αPD1 + 10D1 VS αPD1 + PP4631 P value =<2e−16*** αPD1 + 10D1 VS αPD1 + PP4637 P value = <2e−16*** αPD1 + PP4631VS αPD1 + PP4637 P value = 0.000446***

FIG. 43 . Combination therapy with 10D1 and anti-PD-1 cause anemia,whereas those with either PP4631+anti-PD-1 or PP4637+anti-PD-1 do not.Data shown are hematocrit of 43 day old mice that have received fourtreatments of antibodies on days 11, 14, 17 and 20 at doses of 100μg/mouse/antibodies.

FIGS. 44A-B. Combination therapy with 10D1+anti-PD-1 cause systemic Tcell activation, whereas those with either PP4631+anti-PD-1 orPP4637+anti-PD-1 do not. Data shown are % of CD4 (upper panels) and CD8T cells (lower panels) with phenotypes of naïve (CD44^(lo)CD62L^(hi)),central memory (CD44^(hi)CD62L^(hi)) and effector memory(CD44^(hi)CD62L^(lo)) T cells in either peripheral blood (FIG. 44A) orin the spleen (FIG. 44B). The cells were harvested from 43 day old micethat have received four treatments of antibodies on days 11, 14, 17 and20 at doses of 100 μg/mouse/antibodies.

FIG. 45 . Humanization of L3D10 does not affect binding to immobilizedCTLA4. The capacity of the humanized L3D10 antibodies to bindimmobilized CTLA4 was determined as described in FIG. 36 . X-axisindicates the concentration of anti-CTLA-4 mAbs added into solution.Humanization does not affect binding to immobilized CTLA4 and all 3humanized antibodies demonstrated similar binding to the parentalchimeric L3D10 antibody and 10D1. Similar patterns were observed whenCTLA4-lg was used instead of CTLA-4-his.

FIG. 46 . Humanization further reduces L3D10 binding to soluble CTLA4.The capacity of the humanized L3D10 antibodies to bind soluble CTLA4 wasdetermined as described in FIG. 37 . X-axis indicates the concentrationof anti-CTLA-4 mAbs coated onto ELISA plates. Humanization furtherreduces binding to soluble CTLA4 relative to the parental L3D10 chimericantibody. Similar patterns were observed when CTLA4-Ig was used insteadof CTLA-4-his.

FIGS. 47A-B. PP4631, PP4638 and PP4637 do not block B7-CTLA-4interactions in vitro. FIG. 47A shows blocking of the B7-1-CTLA-4interaction by anti-human CTLA-4 mAbs 10D1, PP4631, PP4637 and L3D10.B7-1Fc was immobilized at the concentration of 0.5 μg/ml. BiotinylatedCTLA4-Fc was added at 0.25 μg/ml along with given doses of antibodies.Data shown are means of duplicate optical density at 405 nM. FIG. 47Bshows blocking of B7-2-CTLA-4 interaction by anti-human CTLA-4 mAbs 10D1and L3D10. As in FIG. 47A, except that B7-2-Fc was immobilized.

FIGS. 48A-B. PP4631 and PP4637 do not block B7-CTLA-4 interactions invivo as demonstrated by their lack of effect on B7-1 and B7-2 expressionon dendritic cells. Summary data on B7-1 (FIG. 48A) and B7-2 (FIG. 48B)levels from 3 mice per group. The B7 levels in the control IgG-treatedgroup are artificially defined as 100%.

FIG. 49 . PP4637, which exhibits the best safety profile in combinationwith anti-PD-1 mAb (see FIG. 42 ), is the most potent in causing tumorrejection based on tumor rejection at the lowest therapeutic doses.Ctla4^(h/m) mice received control IgFc or one of three differentanti-human CTLA4 mab at doses of 30 (−30, solid lines) or 10 (−10,dotted lines) μg per injection at the indicated dates. Tumor sizes weremeasured once every three days. At 10 μg/injection, PP4637 (HL32) is themost efficient in inducing tumor rejection.

FIG. 50 . Humanized antibody purity assessment. Transiently expressedhumanized L3D10 antibodies were purified by Protein A chromatography andsamples from all 3 antibodies was assessed by reducing and non-reducingSDS-PAGE. Purified proteins produced gel bands indicative in size of anantibody molecule under both reducing and non-reducing conditions. The“Flow out” lanes show the protein A column flow through, indicating thatthe majority of the antibody protein adhered to the protein A column.

FIGS. 51A-C. Size Exclusion Chromatography (SE-HPLC) of transientlyexpressed protein. Protein samples for each of the humanized antibodieswere analyzed by SE-HPLC following single step Protein A chromatography.FIG. 51A: antibody PP4631. FIG. 51B: antibody PP4637. FIG. 51C: antibodyPP4638.

FIGS. 52A-C. CE-SDS analysis of transiently expressed protein. Proteinsamples for each of the humanized antibodies were analyzed by CE-SDSfollowing single step Protein A chromatography. Top panels show theresults under non-reduced conditions, and the bottom panels show theresults under reduced conditions. FIG. 52A: antibody PP4631. FIG. 52B:antibody PP4637. FIG. 52C: antibody PP4638.

FIGS. 53A-C. Charge isoform profile and deamidation of the humanizedL3D10 antibodies as determined by capillary isoelectric focusing (cIEF).The level of protein deamidation under high pH stress was determined bycomparing the Humanized L3D10 antibodies before and after high pH stresstreatment over two different time periods (5 hrs and 12.5 hrs) wereanalyzed by cIEF analysis. FIGS. 53A-C show the profiles for antibodiesPP4631, PP4637 and PP4638, respectively.

FIGS. 54A-C. Differential Scanning Calorimetry (DSC) Thermal Analysis ofthe humanized L3D10 antibodies. In order to determine the thermalstability and melting temperatures of the different antibodies, theywere subject to Differential Scanning Calorimetry (DSC) ThermalAnalysis. FIGS. 54A-C show the normalized DSC curves for antibodiesPP4631, PP4637 and PP4638, respectively.

FIG. 55 . Alignment of the human, macaque and mouse CTLA-4 extracellulardomains. The amino acid sequences of the human (Hm)(SEQ ID NO: 73),macaque (Mk) and mouse (Ms) CTLA-4 protein extracellular domains arealigned and the conserved amino acids (relative to the human sequence)are shown with dashes (−). In order to help the alignment, the mousesequence has a deletion and insertion (relative to the human and monkeysequences). The known B7-1Ig binding site is shown in bold and doubleunderlined. The sequences demonstrate that the human and monkeysequences are highly conserved, whereas the mouse sequence has a numberof amino acid differences. Based on this sequence alignment, 11 mutant(M1-M11)(SEQ ID NOS: 40-50) human CTLA-4Fc proteins were designed thatincorporate murine specific amino acids—the amino acids incorporatedinto each mutant (Mut) protein (M1-M11) are shown with lines.

FIGS. 56A-D. Amino acid sequence composition of the WT and mutantCTLA-4Fc proteins. DNA constructs encoding the WT CTLA-4Fc protein (SEQID NO: 39) and 11 mutant proteins (SEQ ID NOS: 40-50) incorporatingmurine Ctla-4 amino acids were designed as shown. The amino acidsequences are for mature proteins, including the IgG1 Fc portion, butnot the signal peptide. The known B7-11g binding site is shown in boldand double-underlined. The replaced murine amino acid residues in themutant are shown lower case in bold italics. The IgG1 Fc portion of theproteins in underlined.

FIGS. 57A-D. Mutation in M11 (AA103-106, YLGI>fcGm) selectively abolishantibody binding to human CTLA-4. Data shown are means of duplicates,depicting the binding of B7-1Fc (FIG. 57A), L3D10 (FIG. 57B), PP4631(FIG. 57C), and PP4637 (FIG. 57D) binding to plate-coated hCTLA4-Fc(open circles), mCTLA4-Fc (filled triangles), M11 (filled circles) andIgG1-Fc (open triangles).

FIG. 58 . Mapping L3D10, PP4631 and PP4637 to an epitope adjacent to theB7-1 binding site in a 3-D structure of the B7-1-CTLA4 complex. The B7-1binding motif is colored in red, while the antibody epitope is coloredin purple. B7-1 is depicted above CTLA4 with a space-filled ribbon,while that of CTLA-4 is depicted as an unfilled ribbon.

FIGS. 59A-C. Amino acid sequence composition of the VVT (SEQ ID NO: 39)and mutant CTLA-4Fc proteins, M12-M17 (SEQ ID NOS: 51-56). DNAconstructs encoding the 6 mutant CTLA-4Fc proteins, M12-M17,incorporating murine Ctla-4 amino acids were designed as shown. Theamino acid sequences are for mature proteins, including the IgG1 Fcportion, but not the signal peptide. The known B7-11g binding site isshown in bold and double-underlined. The replaced murine amino acidresidues in the mutant are shown lower case in bold italics. The IgG1 Fcportion of the proteins in underlined.

FIGS. 60A-C. Mutational analysis reveal distinct binding requirementsfor 10D1 (FIG. 60A), PP4631 (FIG. 60B) and PP4637 (FIG. 60C) to CTLA-4.CTLA-4Fc mutants were coated overnight at 4° C. at 1 μm/ml. Afterblocking with BSA, given concentration of biotinylated anti-CTLA-4 mAbswere added and incubated for 2 hours. After washing away the unboundantibodies, the bound antibodies were detected with HRP-labeledstreptavidin.

FIGS. 61A-B. Therapeutic effect of anti-4-1BB and anti-CTLA-4 antibodiesin both minimal disease (FIG. 61A) and established tumor (FIG. 61B)models. FIG. 61A shows therapy of minimal disease. C57BL/6 mice wereinoculated subcutaneously with 5×10⁵ MC38 cells. On days 2, 9 and 16after tumor cell injection, control hamster and rat IgG, anti-CTLA-4,and/or anti-4-1BB antibodies were injected. Tumor sizes were measured byphysical examination. Data shown are growth kinetics of tumors, witheach line representing tumor growth in one mouse. The sizes presentedare products of long and short diameters of the tumor. FIG. 61B showstherapy of established tumors. As in FIG. 61A, except that therapystarted on day 14 after tumor challenge; all mice had established tumorsranging from 9-60 mm² in size before treatment with mAbs was started.The combined effect of the two antibodies on established tumors has beenrepeated 3 times.

FIG. 62 . CD8 T cells, but not CD4 or NK cells, are essential forantibody-induced tumor rejection. Tumor bearing mice were depleted ofCD4, CD8, or NK cells by three injections of antibodies specific foreither CD4, CD8 or NK1.1 on days 9, 12, and 16 after tumor cellinoculation (*). Therapeutic antibodies (anti-CTLA-4 plus anti-4-1BB)were injected on days 9, 16 and 23 (vertical arrows). Data shown aremeans and SEM of tumor sizes (n=3). P<0.05 for CD8-depleted groupcompared to each of the other groups (†).

FIGS. 63A-B. Combination therapy reduced host response to anti-CTLA-4antibodies. Hamster-anti-mouse-CTLA-4 (FIG. 63A) or rat-anti-mouse-4-1BB(FIG. 63B) antibodies were coated in ELISA plates. Different dilutionsof sera from groups of 5 mice each were added to the plates. Therelative amounts of antibody bound were determined using a secondarystep reagent (biotinylated goat anti-mouse antibodies that were depletedof reactivity to rat and hamster IgG by absorption). Data shown are meanand SEM of optical density at 490 nm. Similar reduction of host antibodyresponse to anti-CTLA-4 and 4-1BB was observed when tumor-free mice weretreated with the same antibodies (data not shown).

FIGS. 64A-B. Combination therapy with anti-4-1BB and L3D10(anti-human-CTLA4) antibody in human CTLA-4 gene knock-in mice. FIG. 64Ashows a therapeutic effect. Human CTLA4 knockin mice were inoculatedwith 5×10⁵ MC38 tumor cells subcutaneously. Two days later, groups of 7mice were treated with rat and mouse IgG, anti-4-1BB and mouse IgG,L3D10 and rat IgG, or L3D10 and anti-4-1BB, as indicated by the arrows.Data shown are mean tumor volume and SEM (n=7). All treatmentssignificantly reduced tumor growth (P<0.001), and the double antibodytreatment group show significantly reduce tumor size in comparison toeither control (P<0.0001) or L3D10 antibody (P=0.0007) or anti-4-1BBantibody treatment (P=0.03). All tumor bearing mice were sacrificed whenthe control IgG-treated group reached early removal criteria. FIG. 64Bshows long-lasting immunity in mice that received combination therapy.Tumor-free mice in the double antibody-treated group developed longlasting immunity to MC38 tumors. At 110 days after the first tumor cellchallenge, the double antibody-treated, tumor-free mice or control naïvemice were challenged with 5×10⁵ tumor cells subcutaneously. Tumor growthwas monitored by physical examination. Note that all of the mice thatrejected the tumors in the first round were completely resistant tore-challenge, while all naïve mice had progressive tumor growth.

DEFINITIONS

As used herein, the term “antibody” is intended to denote animmunoglobulin molecule that possesses a “variable region” antigenrecognition site. The term “variable region” is intended to distinguishsuch domain of the immunoglobulin from domains that are broadly sharedby antibodies (such as an antibody Fc domain). The variable regioncomprises a “hypervariable region” whose residues are responsible forantigen binding. The hypervariable region comprises amino acid residuesfrom a “Complementarity Determining Region” or “CDR” (i.e., typically atapproximately residues 24-34 (L1), 50-56 (L2) and 89-97 (L3) in thelight chain variable domain and at approximately residues 27-35 (H1),50-65 (H2) and 95-102 (H3) in the heavy chain variable domain; ref. 44)and/or those residues from a “hypervariable loop” (i.e., residues 26-32(L1), 50-52 (L2) and 91-96 (L3) in the light chain variable domain and26-32 (H1), 53-55 (H2) and 96-101 (H3) in the heavy chain variabledomain; Ref. 45). “Framework Region” or “FR” residues are those variabledomain residues other than the hypervariable region residues as hereindefined. The term antibody includes monoclonal antibodies, multispecificantibodies, human antibodies, humanized antibodies, syntheticantibodies, chimeric antibodies, camelized antibodies, single chainantibodies, disulfide-linked Fvs (sdFv), intrabodies, and anti-idiotypic(anti-Id) antibodies (including, e.g., anti-Id and anti-anti-Idantibodies to antibodies of the invention). In particular, suchantibodies include immunoglobulin molecules of any type (e.g., IgG, IgE,IgM, IgD, IgA and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 andIgA2) or subclass.

As used herein, the term “antigen binding fragment” of an antibodyrefers to one or more portions of an antibody that contain theantibody's Complementarity Determining Regions (“CDRs”) and optionallythe framework residues that comprise the antibody's “variable region”antigen recognition site, and exhibit an ability to immunospecificallybind antigen. Such fragments include Fab′, F(ab′).sub.2, Fv, singlechain (ScFv),and mutants thereof, naturally occurring variants, andfusion proteins comprising the antibody's “variable region” antigenrecognition site and a heterologous protein (e.g., a toxin, an antigenrecognition site for a different antigen, an enzyme, a receptor orreceptor ligand, etc.). As used herein, the term “fragment” refers to apeptide or polypeptide comprising an amino acid sequence of at least 5contiguous amino acid residues, at least 10 contiguous amino acidresidues, at least 15 contiguous amino acid residues, at least 20contiguous amino acid residues, at least 25 contiguous amino acidresidues, at least 40 contiguous amino acid residues, at least 50contiguous amino acid residues, at least 60 contiguous amino residues,at least 70 contiguous amino acid residues, at least 80 contiguous aminoacid residues, at least 90 contiguous amino acid residues, at least 100contiguous amino acid residues, at least 125 contiguous amino acidresidues, at least 150 contiguous amino acid residues, at least 175contiguous amino acid residues, at least 200 contiguous amino acidresidues, or at least 250 contiguous amino acid residues.

Human, chimeric or humanized antibodies are particularly preferred forin vivo use in humans, however, murine antibodies or antibodies of otherspecies may be advantageously employed for many uses (for example, invitro or in situ detection assays, acute in vivo use, etc.).

A “chimeric antibody” is a molecule in which different portions of theantibody are derived from different immunoglobulin molecules such asantibodies having a variable region derived from a non-human antibodyand a human immunoglobulin constant region. Chimeric antibodiescomprising one or more CDRs from a non-human species and frameworkregions from a human immunoglobulin molecule can be produced using avariety of techniques known in the art including, for example,CDR-grafting (EP 239,400; International Publication No. WO 91/09967; andU.S. Pat. Nos. 5,225,539, 5,530,101, and 5,585,089), veneering orresurfacing (EP 592,106; EP 519,596; 46-48), and chain shuffling (U.S.Pat. No. 5,565,332).

The invention particularly concerns “humanized antibodies”. As usedherein, the term “humanized antibody” refers to an immunoglobulincomprising a human framework region and one or more CDR's from anon-human (usually a mouse or rat) immunoglobulin. The non-humanimmunoglobulin providing the CDR's is called the “donor” and the humanimmunoglobulin providing the framework is called the “acceptor.”Constant regions need not be present, but if they are, they must besubstantially identical to human immunoglobulin constant regions, i.e.,at least about 85-90%, preferably about 95% or more identical. Hence,all parts of a humanized immunoglobulin, except possibly the CDR's, aresubstantially identical to corresponding parts of natural humanimmunoglobulin sequences. A humanized antibody is an antibody comprisinga humanized light chain and a humanized heavy chain immunoglobulin. Forexample, a humanized antibody would not encompass a typical chimericantibody, because, e.g., the entire variable region of a chimericantibody is non-human. One says that the donor antibody has been“humanized,” by the process of “humanization,” because the resultanthumanized antibody is expected to bind to the same antigen as the donorantibody that provides the CDR's. For the most part, humanizedantibodies are human immunoglobulins (recipient antibody) in whichhypervariable region residues of the recipient are replaced byhypervariable region residues from a non-human species (donor antibody)such as mouse, rat, rabbit or a non-human primate having the desiredspecificity, affinity, and capacity. In some instances, Framework Region(FR) residues of the human immunoglobulin are replaced by correspondingnon-human residues. Furthermore, humanized antibodies may compriseresidues which are not found in the recipient antibody or in the donorantibody. These modifications are made to further refine antibodyperformance. In general, the humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the hypervariable regionscorrespond to those of a non-human immunoglobulin and all orsubstantially all of the FRs are those of a human immunoglobulinsequence. The humanized antibody optionally also will comprise at leasta portion of an immunoglobulin constant region (Fc), typically that of ahuman immunoglobulin that immunospecifically binds to an Fc.gamma.RIIBpolypeptide, that has been altered by the introduction of amino acidresidue substitutions, deletions or additions (i.e., mutations).

DETAILED DESCRIPTION

An antibody against human CTLA4 protein, Ipilimumab, has been shown toincrease survival of cancer patients, either as the onlyimmunotherapeutic agent or in combination with other therapeutic agentssuch as, for example without limitation, an anti-PD-1 antibody (13-15).However, the therapeutic effect is associated with significant adverseeffects (13-18). There is a great need to develop novel anti-CTLA4antibodies to achieve better therapeutic effect and/or less autoimmuneadverse effect. The inventors have discovered an anti-CTLA4 antibodythat, surprisingly, can be used to induce cancer rejection while alsoreducing autoimmune adverse effects associated with immunotherapy.

Provided herein are antibody compositions of matter and antigen-bindingfragments thereof. The invention further concerns the embodiment of suchmolecules wherein the molecule is a monoclonal antibody, a humanantibody, a chimeric antibody or a humanized antibody.

In detail, the invention provides a molecule, comprising anantigen-binding fragment of an antibody that immunospecifically binds toCTLA4, and in particular human CTLA4, preferably expressed on thesurface of a live cell at an endogenous or transfected concentration.The invention particularly concerns the embodiment of such a moleculewherein the antigen-binding fragment binds to CTLA4, and wherein thelive cell is a T cell.

The present invention relates to antibodies and their antigen-bindingfragments that are capable of immunospecifically binding to CTLA4. Insome embodiments such molecules are additionally capable of blocking thebinding of B7.1 and B7.2 to CTLA4.

The invention further concerns the embodiment of such molecules whereinthe molecule is a monoclonal antibody, a human antibody, a chimericantibody or a humanized antibody. The invention includes the embodimentswherein such antibodies are monospecific, bispecific, trispecific ormultispecific.

The invention further concerns the embodiment of such molecules orantibodies which binds to CTLA4, and wherein the antigen-bindingfragment thereof comprises six CDRs, wherein the CDRs comprise the CDRsof anti-CTLA4 antibody L3D10. Specifically, the antibody comprises thethree light chain and the three heavy chain CDRs of anti-CTLA4 antibodyL3D10.

The invention further concerns the embodiment of the above-describedantibodies, wherein the antibody is detectably labeled or comprises aconjugated toxin, drug, receptor, enzyme, receptor ligand.

The invention further concerns a pharmaceutical composition comprising atherapeutically effective amount of any of the above-described antibodycompositions, and a physiologically acceptable carrier or excipient.Preferably, compositions of the invention comprise a prophylactically ortherapeutically effective amount of humanized antibodies of theinvention and a pharmaceutically acceptable carrier

In a specific embodiment, the term “pharmaceutically acceptable” meansapproved by a regulatory agency of the Federal or a state government orlisted in the U.S. Pharmacopeia or other generally recognizedpharmacopeia for use in animals, and more particularly in humans. Theterm “carrier” refers to a diluent, adjuvant (e.g., Freund's adjuvant(complete and incomplete), excipient, or vehicle with which thetherapeutic is administered. Such pharmaceutical carriers may be sterileliquids, such as water and oils, including those of petroleum, animal,vegetable or synthetic origin, such as peanut oil, soybean oil, mineraloil, sesame oil and the like. Water is a preferred carrier when thepharmaceutical composition is administered intravenously. Salinesolutions and aqueous dextrose and glycerol solutions can also beemployed as liquid carriers, particularly for injectable solutions.Suitable pharmaceutical excipients include starch, glucose, lactose,sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate,glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol,propylene, glycol, water, ethanol and the like. The composition, ifdesired, may also contain minor amounts of wetting or emulsifyingagents, or pH buffering agents. These compositions may take the form ofsolutions, suspensions, emulsion, tablets, pills, capsules, powders,sustained-release formulations and the like.

Generally, the ingredients of compositions of the invention may besupplied either separately or mixed together in unit dosage form, forexample, as a dry lyophilized powder or water free concentrate in ahermetically sealed container such as an ampoule or sachette indicatingthe quantity of active agent. Where the composition is to beadministered by infusion, it can be dispensed with an infusion bottlecontaining sterile pharmaceutical grade water or saline. Where thecomposition is administered by injection, an ampoule of sterile waterfor injection or saline may be provided so that the ingredients may bemixed prior to administration.

The compositions of the invention may be formulated as neutral or saltforms. Pharmaceutically acceptable salts include, but are not limitedto, those formed with anions such as those derived from hydrochloric,phosphoric, acetic, oxalic, tartaric acids, etc., and those formed withcations such as those derived from sodium, potassium, ammonium, calcium,ferric hydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol,histidine, procaine, etc.

The invention further concerns the use of the antibody compositionsdescribed here and pharmaceutical compositions thereof for theupregulation of immune responses. Up-modulation of the immune system isparticularly desirable in the treatment of cancers and chronicinfections, and thus the present invention has utility in the treatmentof such disorders. As used herein, the term “cancer” refers to aneoplasm or tumor resulting from abnormal uncontrolled growth of cells.As used herein, cancer explicitly includes leukemias and lymphomas. Theterm refers to a disease involving cells that have the potential tometastasize to distal sites.

Accordingly, the methods and compositions of the invention may also beuseful in the treatment or prevention of a variety of cancers or otherabnormal proliferative diseases, including (but not limited to) thefollowing: carcinoma, including that of the bladder, breast, colon,kidney, liver, lung, ovary, pancreas, stomach, cervix, thyroid and skin;including squamous cell carcinoma; hematopoietic tumors of lymphoidlineage, including leukemia, acute lymphocytic leukemia, acutelymphoblastic leukemia, B-cell lymphoma, T-cell lymphoma, Berkettslymphoma; hematopoietic tumors of myeloid lineage, including acute andchronic myelogenous leukemias and promyelocytic leukemia; tumors ofmesenchymal origin, including fibrosarcoma and rhabdomyoscarcoma; othertumors, including melanoma, seminoma, tetratocarcinoma, neuroblastomaand glioma; tumors of the central and peripheral nervous system,including astrocytoma, neuroblastoma, glioma, and schwannomas; tumors ofmesenchymal origin, including fibrosarcoma, rhabdomyosarcoma, andosteosarcoma; and other tumors, including melanoma, xenodermapegmentosum, keratoactanthoma, seminoma, thyroid follicular cancer andteratocarcinoma. It is also contemplated that cancers caused byaberrations in apoptosis would also be treated by the methods andcompositions of the invention. Such cancers may include, but are not belimited to, follicular lymphomas, carcinomas with p53 mutations, hormonedependent tumors of the breast, prostate and ovary, and precancerouslesions such as familial adenomatous polyposis, and myelodysplasticsyndromes. In specific embodiments, malignancy or dysproliferativechanges (such as metaplasias and dysplasias), or hyperproliferativedisorders, are treated or prevented by the methods and compositions ofthe invention in the ovary, bladder, breast, colon, lung, skin,pancreas, or uterus. In other specific embodiments, sarcoma, melanoma,or leukemia is treated or prevented by the methods and compositions ofthe invention.

In another embodiment of the invention, the antibody compositions andantigen binding fragments thereof can be used with other anti-tumortherapies, including but not limited to, current standard andexperimental chemotherapies, hormonal therapies, biological therapies,immunotherapies, radiation therapies, or surgery. In some embodiments,the molecules of the invention may be administered in combination with atherapeutically or prophylactically effective amount of one or moreagents, therapeutic antibodies or other agents known to those skilled inthe art for the treatment and/or prevention of cancer, autoimmunedisease, infectious disease or intoxication. Such agents include forexample, any of the above-discussed biological response modifiers,cytotoxins, antimetabolites, alkylating agents, antibiotics, oranti-mitotic agents, as well as immunotherapeutics.

In preferred embodiment of the invention, the antibody compositions andantigen binding fragments thereof can be used with other anti-tumorimmunotherapies. In such an embodiment the molecules of the inventionare administered in combination with molecules that disrupt or enhancealternative immunomodulatory pathways (such as TIM3, TIM4, OX40, CD40,GITR, 4-1-BB, B7-H1, PD-1, B7-H3, B7-H4, LIGHT, BTLA, ICOS, CD27 orLAG3) or modulate the activity of effecter molecules such as cytokines(e.g., IL-4, IL-7, IL-10, IL-12, IL-15, IL-17, GF-beta, IFNg, Flt3,BLys) and chemokines (e.g., CCL21) in order to enhance theimmunomodulatory effects. Specific embodiments include a bi-specificantibody comprising the anti-CTLA4 antibody compositions describedherein and anti-PD-1 (pembrolizumab (Keytruda) or Nivolumab (Opdivo)),anti-B7-H1 (atezolizumab (Tecentriq) or durvalumab), anti-B7-H3,anti-B7-H4, anti-LIGHT, anti-LAG3, anti-TIM3, anti-TIM4 anti-CD40,anti-OX40, anti-GITR, anti-BTLA, anti-CD27, anti-ICOS or anti-4-1BB. Inyet another embodiment, the molecules of the invention are administeredin combination with molecules that activate different stages or aspectsof the immune response in order to achieve a broader immune response. Inmore preferred embodiment, the antibody compositions and antigen bindingfragments thereof are combined with anti-PD-1 or anti-4-1BB antibodies,without exacerbating autoimmune side effects.

Another embodiment of the invention includes a bi-specific antibody thatcomprises an antibody that binds to CTLA4 bridged to an antibody thatbinds another immune stimulating molecules. Specific embodiments includea bi-specific antibody comprising the anti-CTLA4 antibody compositionsdescribed herein and anti-PD-1, anti-B7-H1, anti-B7-H3, anti-B7-H4,anti-LIGHT, anti-LAG3, anti-TIM3, anti-TIM4 anti-CD40, anti-OX40,anti-GITR, anti-BTLA, anti-CD27, anti-ICOS or anti-4-1BB. The inventionfurther concerns of use of such antibodies for the treatment of cancer.

Methods of administering the antibody compositions of the inventioninclude, but are not limited to, parenteral administration (e.g.,intradermal, intramuscular, intraperitoneal, intravenous andsubcutaneous), epidural, and mucosal (e.g., intranasal and oral routes).In a specific embodiment, the antibodies of the invention areadministered intramuscularly, intravenously, or subcutaneously. Thecompositions may be administered by any convenient route, for example,by infusion or bolus injection, by absorption through epithelial ormucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa,etc.) and may be administered together with other biologically activeagents. Administration can be systemic or local.

Yet another embodiment of the invention concerns monitoring the blockingeffects of anti-CTLA4 antibodies in vivo by monitoring the expressionlevels of B7.1 and B7.2 on immune cells such as antigen presenting cells(APCs). CTLA4 is expressed predominately among the Treg where itsuppresses autoimmune diseases by down-regulating B7-1 and B7-2expression on APCs such as dendritic cells. Therefore, upregulation ofB7 molecules, B7.1 and B7.2, can be as readouts for the in vivo blockadeof B7-CTLA4 interactions. In a specific embodiment, peripheral orintra-tumoral immune cells are removed from the subject before and afteranti-CTLA4 treatment and assayed ex vivo for a reduction in the level ofB7.1 and/or B7.2 on the surface of the immune cell, wherein the presenceof blocking anti-CTLA4 antibodies prevents B7.1/B7.2 binding byendogenous CTLA4, which in turn prevents the downregulation of B7.1 andB7.2, resulting in a net increase in B7.1/B7.2 expression. In apreferred embodiment the level of B7.1 and B7.1 is measured on antigenpresenting cells. In a most preferred embodiment the level of B7.1 andB7.1 is measured on dendritic cells.

In a further embodiment, the change (reduction) in B7.1 and B7.2 onimmune cells following anti-CTLA4 treatment is used as a biomarker formeasuring the biological activity of anti-CTLA4 antibodies in vivo andmonitoring patent responses to anti-CTLA4 treatment by measuring thelevel B7.1 and/or B7.2 expression on immune cells, and comparing thelevel of expression before and after treatment. In a preferredembodiment the level of B7.1 and/or B7.2 expression is monitored overtime during a course of anti-CTLA4 therapy.

EXAMPLES Example 1. Generation of Chimeric Anti-CTLA4 Antibody

Using human CTLA4 gene knock-in mice and hu-PBL-Scid mice, it waspreviously demonstrated that mouse anti-human CTLA4 antibodies reducedtumor growth, and identify L3D10 as the most effective among the panelof mAbs tested. However, none of the antibodies obtained was able toachieve complete tumor rejection, even when used at relatively highdoses of (>10 mg/kg) and before formation of palpable tumors (as earlyon day 2) after tumor cell challenge (19-21).

Since the mouse antibodies were of IgG1 subclass that does not havestrong antibody-dependent cellular cytotoxicity (ADCC), and since ADCCmaybe involved in tumor rejection, the Fc of the mAb was modified inseveral ways, to achieve better immunotherapeutic effect. First, mouseIgG1, which is weak in ADCC, was replaced to produce a chimeric antibodywith human IgG1, which has strong ADCC activity. Second, based on knownart in the literatures (22), three mutations (S298A, E333A and K334A)were introduced in the CH to increase ADCC activity. Third, threemutations (M252Y, S254T and T256E) were introduced to increase thehalf-life of the antibody in vivo (23). The design of the new chimericantibody is depicted in FIG. 1 , left panel.

To engineer the antibody, the variable regions of L3D10 hybridoma werefirst identified through DNA sequencing using standard methods known inthe art. The nucleotide sequences were translated into amino acidslisted in SEQ ID NO: 1 and SEQ ID NO: 2. The normal human IgG1 Fcsequence and the mutant Fc sequence are disclosed in SEQ ID NO: 3 andSEQ ID NO: 4, respectively. The amino acid and codon optimizednucleotide sequences of heavy and light chain sequences are disclosed inSEQ ID NOS: 5-8.

DNA corresponding to SEQ ID NO: 5 and SEQ ID NO: 7 were synthesized andinserted into expression vectors, and the vectors were transfected withthe designed sequence into HEK293 cells. Briefly, HEK293 cells wereseeded in a shake flask one day before transfection, and were grownusing serum-free chemically defined media. The DNA expression constructswere transiently transfected into 0.5 liter of suspension HEK293 cellsusing standard operating procedure for transient transfection. After 20hours, cells were sampled to obtain the viability and viable cell count,and titer was measured (Octet QKe, ForteBio). Additional readings weretaken throughout the transient transfection production runs. The culturewas harvested at day 5. The conditioned media for L3D10 was harvestedand clarified from the transient transfection production run bycentrifugation and filtration. The supernatant was run over a Protein Acolumn and eluted with a low pH buffer. Filtration using a 0.2 μmmembrane filter was performed before aliquoting. After purification andfiltration, the protein concentration was calculated from the OD280 andthe extinction coefficient. A total of 43.2 mg of Ig proteins wereobtained from one round of transfection.

Example 2. Chimeric L3D10 Antibody Binding Sites Only Partially Overlapwith 10D1

In the clinic, the anti-CTLA4 antibody, Ipilimumab, has been shown toimprove the survival of cancer patients but induce significantautoimmune adverse effect. In order to evaluate the comparative bindingsites of the chimeric L3D10 antibody and 10D1, binding to CTLA4 and theability of the antibodies to compete for binding to CTLA4 were compared.While both antibodies bind to immobilized CTLA4 proteins at comparableefficiency (FIG. 2 ), 10D1 does not completely block chimeric L3D10binding to CTLA4 (FIG. 3 ). As expected, unlabeled L3D10 completelyblocks labeled L3D10 binding, indicating that the antibody binding sitesof L3D10 and 10D1 only partially overlap.

Example 3. More Efficient Blockade CTLA4:B7.1 and CTLA4:B7.2Interactions by Chimeric L3D10 Antibody than by 10D1

It has been reported that anti-human CTLA4 mAb, 10D1, can block B7-CTLA4interaction if soluble B7-1 and B7-2 was used to interact withimmobilized CTLA4 (49). Since B7-1 and B7-2 function as cell surfaceco-stimulatory molecules, we evaluated the ability of anti-CTLA4antibodies to block the B7-CTLA4 interaction using immobilized B7-1 andB7-2. Using a competitive ELISA assay format, the abilities of L3D10 and10D1 to block binding of the CTLA4 fusion protein, CTLA4-Ig, to bothplate-immobilized- and cell membrane-expressed B7.1 and B7.2. For theseexperiments a chimeric anti-human CTLA4-mAb with an affinity (2.3 nM)that is similar to 10D1 (4 nM) (49) was used. For the plate immobilizedassays, B7.1 Fc or B7.2Fc were coated onto the ELISA plate at 1 μg/mlover night at 4° C. or 2 hours at 37° C. Biotinylated CTLA4-Fc weremixed with given concentrations of either B7.1-Fc, 10D1 or chimericL3D10. The amounts of the CTLA4-Fc bound to B7.1 on the plate isdetermined using horse-radish peroxidase-conjugated streptavidin. Asshown in FIG. 4 , while chimeric L3D10, B7.1 Fc and CTLA4-Fc allefficiently blocked CTLA4-Fc:B7.1 interaction, two separate materiallots of 10D1 failed to block the interaction. L3D10 shows significantblocking of plate-immobilized B7.1 binding at concentrations as low as0.2 μg/ml, achieving 50% inhibition (IC₅₀) at around 3 μg/ml. Similarly,L3D10 blocked binding of CTLA4-Fc binding to plate immobilized B7.2 withan IC₅₀ of 0.03 μg/ml, whereas 10D1 from two different material lotsdisplayed minimal blocking with an IC₅₀ of approximately 200 μg/ml (FIG.5 ). However, consistent with the previous report (49), antibody 10D1potently inhibited B7-1-CTLA4 interaction in the reverse experiment whenplate immobilized CTLA4 is used to interact with soluble B7-1 (FIG. 6 ).

For the cell membrane protein binding experiments, when B7.1 isexpressed on the surface of CHO cells, L3D10 blocks binding of CTLA4-Fcbut 10D1 from two different material lots did not, even when used at 512μg/ml (FIG. 7 ). While much less potent than L3D10, high doses of 10D1achieved approximately 25% blocking between human CTLA4 and mouse B7-1(FIG. 8 ). For B7.2 expressed on the CHO cell surface, L3D10 was againblocking whereas 10D1 was only partially blocking, with less than 50%inhibition observed even when 10D1 was used at 512 μg/ml (FIG. 9 ).

A potential caveat is that biotinylation may have affected binding of10D1 to CTLA4-Fc. To address this issue, we compared binding of L3D10and 10D1 to biotinylated CTLA4-Fc used in the blocking studies. As shownin FIG. 10 , 10D1 is more effective than L3D10 in binding thebiotinylated CTLA4-Fc. Therefore, the failure in blockade by 10D1 wasnot due to insufficient binding to biotinylated CTLA4-Fc. A similarpattern is observed when polyhistidine-tagged CTLA4 was used to interactwith human B7-1 transfected CHO cells (FIG. 11 ). Taken together, ourdata suggest that ability of antibody 10D1 to block B7-CTLA4 interactionis highly dependent on the assay employed, with minimal to no detectableblocking activity if B7-1 and B7-2 are immobilized, while antibody L3D10is a robust blocker for B7-CTLA4 interaction regardless of whether theB7 protein is immobilized.

Example 4. Chimeric L3D10 Antibody is More Efficient than UnmodifiedL3D10 in Causing Tumor Rejection

It was previously reported that mouse L3D10 failed to cause completeremission of MC38 tumors, even though significant delays were observed(19, 20). To determine if chimeric L3D10 can cause complete remission insyngeneic mice, 1×10⁶ MC38 tumor cells were transplanted into syngeneicC57BL/6 mice. One week later, when the tumor reaches around 5 mm indiameter, mice were treated with either control IgG or chimeric L3D10mAb at a dose that is only half of what was used in the previous studieswith the mouse L3D10. As shown in FIG. 12 , despite possibleimmunogenity of the human Ig sequence, it was found that the chimericL3D10 caused complete remission in all mice tested. Since the treatmentwas initiated when large tumor burdens have been established, which ismuch more difficult than when tumors were not palable (19), theseexperiments show that chimeric L3D10 is more efficient than unmodifiedL3D10.

Example 5. Chimeric L3D10 Antibody has Equivalent Activity as 10D1 inCausing Tumor Rejection

The availability of human CTLA4 gene knockin mice (20) provided with anunprecedented opportunity to test biological activity of the chimericanti-human CTLA-4 antibody with clinically used anti-CTLA-4 mAb, 10D1.In this humanized mouse model, a CTLA4 gene encoding a product with 100%identity to human CTLA-4 protein is expressed under the control ofendogenous mouse Ctla4 locus When the anti-tumor activity of thechimeric L3D10 and 10D1 were directly compared in the MC38 tumor modelin human CTLA4-knockin mice, it is clear that both antibodies werecomparable in causing tumor rejection, whereas the tumors grewprogressively in IgG control group. FIG. 13 shows the results ofantibody treatment on tumor size from duplicate experiments.

An interesting question is whether anti-CTLA-4 mAbs need to interactwith all CTLA-4 (i.e. achieve target saturation) in order to exertimmunotherapeutic effect. F1 mice from CTLA4^(h/h) and CTLA4^(m)/m miceexpresses both mouse and human CTLA-4 protein in a co-dominant manner.Interestingly, as shown in FIG. 14 , both chimeric L3D10 and 10D1effectively induced tumor rejection, even though approximately 50% ofthe CTLA-4 protein (i.e. the murine version of the protein) cannot bebound by anti-human CTLA-4 mAbs. Importantly, L3D10 is moretherapeutically effective than 10D1 in this setting i.e. when gene dosesare limited (P<0.05).

Previous studies have demonstrated that anti-mouse Ctla-4 mAbs cannotinduce rejection of melanoma cell line B16-F1 without combination withother therapeutic modalities. Therefore, the anti-tumor effect of thechimeric L3D10 and 10D1 antibodies was also tested using this morechallenging B16 tumor model in the human CTLA4 knockin mice. As shown inFIG. 15 , whereas neither L3D10 nor Ipilimumab were capable of causingrejection of established tumors, both cause statistically significantretardation of tumor growth, while the differences between differentantibodies are not statistically significant.

Example 6: CTLA4 Blocking In Vivo

CTLA4 is expressed predominately among the Treg where it suppressesautoimmune diseases by down-regulating B7-1 and B7-2 expression ondendritic cells (50). Since targeted mutation of Ctla4 (50) andtreatment with blocking anti-CTLA4 mAb (51) upregulated expression ofB7-1 and B7-2 on dendritic cells, it has been suggested thatphysiological function of CTLA4 on Treg is to down-regulate B7 on DC.Therefore, upregulation of B7 was used as a readout for the in vivoblockade of B7-CTLA4 interactions and developed an assay using T cellsfrom the Ctla4^(h/h) mice which had homozygous knockin of the humanCTLA4 gene.

As outlined in FIG. 16 , surface expressed B7.1 or B7.2 binds CTLA4 onthe surface of T cells, which leads to a downregulation in B7.1 and B7.2expression. However, binding of blocking anti-CTLA4 antibodies preventsB7.1/B7.2 binding, which prevents the downregulation of B7.1 and B7.2,resulting in a net increase in B7.1/B7.2 expression. However, withchimeric T cells expressing both human and mouse CTLA4, antibodies thatbind human CTLA4 do not prevent B7.1/B7.2 binding to the murine CTLA4,which restores B7.1/B7.2 inhibition.

CTLA4 humanized mice that express the CTLA4 gene with 100% identify tohuman CTLA4 protein under the control of endogenous mouse Ctla4 locushas been described (20). The homozygous knock-in mice (CTLA4^(h/h)) werebackcrossed to C57BL/6 background for at least 10 generations.Heterozygous mice (CTLA4^(h/m)) were produced by crossing theCTLA4^(h/h) mice with WT BALB/c mice.

To test clinically proven therapeutic anti-CTLA4 mAb, 10D1, we injectedvery high doses of anti-CTLA4 mAb (500 μg/mouse, which is roughly 25mg/kg or 8-times the highest dose used in the clinic) into Ctla4^(h/)hor Ctla4^(m/h) mice and harvested spleen cells to measure levels of B7-1and B7-2 on Cd11c^(hi) DC at 24 hours after injection (FIGS. 17A-B). Asshown FIGS. 17C-E, in comparison to Ctla4^(h/h) mice that received humanIgG1-Fc, DC from chimeric L3D10 treated mice had a statisticallysignificant increase in B7.1 expression in T cells expressing humanCTLA4 but not in T cells expressing both human and mouse CTLA4. Similarresults were seen for B7.2 as shown in FIGS. 17C-E. The magnitude ofupregulation in B7-2 is comparable to what was achieved using a blockinganti-CTLA4 mAb in human Treg-DC co-culture (66).

To further confirm the specificity of the in vivo assay, we tested ifL3D10 can upregulate B7 in Ctla4^(m/h) mice in which mouse and humanCTLA4 are expressed co-dominantly. Since at least 50% of the CTLA4 doesnot bind to anti-human CTLA4 antibodies, it is expected that they wouldbe less potent in blocking B7-CTLA4 interaction. Indeed, neitherantibody caused upregulation of B7-1 and B7-2 on DC from Ctla4^(m/h)mice (FIG. 17C, D, F). The complete lack of blockade by L3D10 in theCtla4^(m/h) mice suggests that CTLA4 encoded by the mouse allele, whichdoes not bind to L3D10 (FIG. 18 ), is sufficient to down-regulate B7expression. Thus, our data demonstrated that at doses that are at least8-times higher than the highest dose used in clinic, 10D1 does not blockB7-CTLA4 interaction when B7 are either immobilized on plate or anchoredon cell membrane, both in vivo and in vitro.

The complete lack of blockade by L3D10 in the Ctla4^(m/h) mice suggeststhat CTLA4 encoded by the mouse allele, which does not bind to L3D10(FIG. 18 ), is sufficient to down-regulate B7 expression. In contrast,10D1 did not increase B7.1 or B7.2 expression. According to the model,this suggests that L3D10 blocks CTLA4 activity in vivo whereas 10D1 doesnot.

However, despite these apparent differences in blocking activity, bothL3D10 and 10D1 display strong anti-tumor activity against the MC38 modelin chimeric CTLA4^(m/h) mice, as shown in FIG. 19 . While the tumor grewprogressively in the control Ig-treated mice, complete rejection wasachieved by either anti-CTLA4 mAb. In multiple experiments, the twoantibodies are comparable in causing tumor rejection. In another tumormodel, B16 melanoma, both antibodies induced similar retardation oftumor growth, although complete rejection was not achieved by eitherantibody (FIG. 20 ).

Example 7: Anti-Tumor Effects are Associated with Intra-Tumoral TregDepletion

Immune regulation in vivo results from a balance between immune cellactivation and immune checkpoints. In particular, regulatory T cells(Tregs) are a subpopulation of T cells which regulate the immune system,maintain tolerance to self-antigens, and abrogate autoimmune disease.Recent studies have demonstrated that therapeutic efficacy of anti-mouseCTLA4 mAb is affected by the Fc subclass and host Fc receptor, which inturn affect antibody-dependent cytotoxicity of Treg selectively withintumor microenvironment (52, 53). As differential CTLA4 blocking activityin vivo does not appear to translate to differences in anti-tumoractivity, we attempted to establish the mechanism of action(s) by whichthe anti-tumor occurs and looked at Tregs within the tumormicroenvironment. To do this, we sacrificed MC38 tumor-bearing micebefore the rejections were completed (FIG. 21 ) and analyzed thefrequency of Treg in Ctla4^(h/h) knockin mice that received control Ig,10D1 or L3D10. While neither antibody reduces Treg in the spleen (FIG.22C), both reduced Treg in the tumor microenvironment (FIG. 22E,).Interestingly, 10D1 but not L3D10 expanded Treg in the spleen. Expansionof Treg in the spleen by 10D1 recapitulates a clinical finding thatIpilimumab increased FOXP3 expression by the peripheral blood leukocytes(54). Since the blocking and non-blocking antibodies are comparable indepletion of Treg in the tumor microenvironment, blockade of B7-CTLA4interaction does not contribute to Treg depletion. Since 10D1 does notblock B7-CTLA4 interaction in vivo and yet confer therapeutic effect inthe Ctla4^(h/h) mice and in melanoma patients, blockade of thisinteraction is not required for its therapeutic effect. Furthermore,since two mAbs with drastically different blocking effect havecomparable therapeutic effect and selective Treg depletion in tumormicroenvironment, blocking CTLA4-B7 interaction does not enhancetherapeutic effect of an antibody.

To substantiate this observation, we tested the therapeutic effect ofthe two anti-CTLA4 mAbs in the Ctla4^(m/h) mice in which the anti-humanCTLA4 mAbs can bind to at maximal of 50% of CTLA4 molecules and in whichneither antibody can block B7-CTLA4 interaction to achieve upregulationof B7 on dendritic cells (FIG. 16 ). Again, both antibodies cause rapidrejection of the MC38 tumors, although L3D10 is somewhat more effectivethan 10D1 (FIG. 22B). Correspondingly, both antibodies selectivelydepleted Treg in tumor microenvironment (FIGS. 22D and 21F). Thesegenetic data further demonstrated the irrelevance of CTLA4 blockade intumor rejection and local Treg depletion and thus refute the prevailinghypothesis that anti-CTLA4 mAb induce cancer immunity through blockingB7-CTLA4 interaction (10).

Example 8. Evaluation of Blocking Activities of Commonly Used Anti-MouseCTLA4 mAbs 9H10 and 9D9

The concept that CTLA4 is a cell-intrinsic negative regulator for T cellregulation was proposed based on stimulatory effect of both intact andFab of two anti-mouse CTLA4 mAbs (30, 31), 4F10 and 9H10, although nodata were presented to demonstrate that these antibodies block B7-CTLA4interaction. More recently, a third anti-mouse CTLA4 mAb, 9D9, wasreported to have therapeutic effect in tumor bearing mice and causelocal depletion of Treg in tumor microenvironment (52). We therefore setout to test all three commercially available anti-mouse CTLA4 mAbs thathad been shown to induce tumor rejection for their ability to blockB7-CTLA4 interaction under physiologically relevant configurations. As afirst test, we used increasing amounts of anti-CTLA4 mAbs (up to 2,000fold molar excess over CTLA4-Fc) to block binding of biotinylatedCTLA4-Fc to plate-immobilized B7-1 and B7-2. As shown in FIG. 23A,anti-mouse CTLA4 mAb 9H10 did not block the B7-1-CTLA4 interaction evenat the highest concentration tested, although a modest blocking wasobserved when 9D9 was used at very high concentrations. While mAb 9D9effectively blocked the B7-2-CTLA4 interaction, 9H10 failed to do so(FIG. 23B). Interestingly, while 9D9 shows strong binding to solubleCTLA4-Fc, 9H10 showed poor binding (FIG. 23 c ), even though it is morepotent than 9D9 in binding immobilized mouse CTLA4-Fc (FIG. 23D). Sincelack of any blocking activity by 9H10 in this assay may simply reflectits poor binding to soluble CTLA4-Fc, we again used up-regulation ofB7-1 and B7-2 on dendritic cells in WT mice (CTLA4^(m)/m) to measure invivo blocking of B7-CTLA4 interaction. As shown in FIGS. 23E and F, 9H10did not upregulate B7-1 expression on DC, while 9D9 increased B7-1 levelby 15% (P<0.05). Interestingly, while 9D9 clearly upregulated B7-2 onDC, 9H10 failed to do so. Therefore, 9H10, the first and mostextensively studied tumor immunotherapeutic anti-CTLA4 mAb does notblock B7-CTLA4 interaction. Therefore, blocking B7-CTLA4 interactiondoes not contribute to induction of anti-tumor immunity by anti-mouseCTLA4 mAbs. Since both mAbs show comparable immunotherapeutic effect andcomparable deletion of Treg in the tumor microenvironment (52), localdeletion of Treg, rather than blockade of B7-CTLA4 interaction, providesa unifying explanation for therapeutic effect of anti-mouse CTLA4 mAbs.Interestingly, while 4F10 blocked B7-CTLA4 interaction in vitro, itfailed to induce upregulation of B7 on DC in vivo (FIG. 24 ).

Taken together, we have demonstrated that clinically proven therapeuticanti-human CTLA4 mAb (10D1) and two anti-mouse CTLA4 mAbs (9H10 and4F10) confers immunotherapeutic effect without blocking B7-CTLA4interaction under physiologically relevant conditions. Furthermore, suchblockade was not necessary for tumor rejection even for the mAb (L3D10)that can potently block B7-CTLA4 interaction. Since the therapeuticeffect is substantially the same for antibodies with 1000-folddifferences in blocking B7-CTLA4 interaction, such blockade does notcontribute to cancer therapeutic effect of the anti-CTLA4 mAbs. Thesedata refute the hypothesis that anti-CTLA4 mAb confers immunotherapeuticeffect through checkpoint blockade (55). By refuting the prevailinghypothesis, our data suggest that the therapeutic effect of anti-CTLA4mAb cannot be optimized by improving the blocking activities of theanti-CTLA4 mAbs. In this context, it is particular interest to note thatTremelimumab, which is superior in blocking B7-CTLA4 interaction (56),did not reach clinical endpoint in a phase III clinical trial (57).Meanwhile, by demonstrating strong correlation between tumor rejectionof local Treg depletion and by refuting the involvement of blockade ofB7-CTLA4 interaction in tumor immunity, our work favor the hypothesisthat local deletion of Treg within the tumor environment is the mainmechanism for therapeutic anti-CTLA4 mAb, and hence suggest newapproaches to develop next generation of anti-CTLA4 mAb for cancerimmunotherapy.

Finally, accumulating genetic data in the mice suggest that the originalconcept (30, 31) that CTLA4 negatively regulates T cell activation andthat such regulation was achieved through SHP-2 (58, 59) may need to berevisited (60). Thus, while the severe autoimmune diseases in theCtla4^(−/−) mice have been used to support the notion of CTLA4 as acell-intrinsic negative regulator for T cell activation (61, 62), atleast three lines of genetic data have since emerged that are notconsistent with this view. First, lineage-specific deletion of the Ctla4gene in Treg but not in the effector T cells is sufficient torecapitulate the autoimmune phenotype observed in mice with germlinedeletion of the Ctla4 gene (50). These data suggest that theautoimmunity in the Ctla4^(−/−) mice was not due to lack ofcell-intrinsic negative regulator CTLA4 in effector T cells. Second, inchimera mice consisting of both WT and Ctla4^(−/−) T cells, theautoimmune phenotype was prevented by co-existence of WT T cells (63).These data again strongly argue that autoimmune diseases were not causedby lack of cell-intrinsic negative regulator. The lack of cell-intrinsicnegative regulator effect is also demonstrated by the fact that in thechimera mice, no preferential expansion of Ctla4^(−/−) T cells wasobserved during viral infection (64). Third, T-cell specific deletion ofShp2, which was proposed to be mediating negative regulation of CTLA4(58, 59), turned out to reduce rather than enhance T cell activation(65). In the context of these genetic data reported since the proposalof CTLA4 as negative regulator for T cell activation, our data reportedherein call for a reappraisal of CTLA4 checkpoint blockade in cancerimmunotherapy.

Example 9. Chimeric L3D10 Demonstrates Reduced Immune Adverse Eventswhen Used in Combination with Other Immunotherapeutic Antibodies

Recent clinical studies have revealed that combination therapy betweenanti-PD-1 and anti-CTLA4 mAb further increase the survival of end-stagemelanoma patients. However, 55% of the patients that received thecombination therapy developed grades 3 and 4 immune related adverseevents (irAEs). It is therefore critical to develop antibodies with lesstoxicity. We have developed an in vivo model that recapitulates theirAEs associated with the combination therapy of anti-CTLA-4 andanti-PD-1 mAbs observed in the clinic. In this model we treated humanCTLA4 gene knockin mice (CTLA4^(h/h)) during the perinatal period withhigh doses of anti-PD-1 and anti-CTLA-4 mAbs. We found that while theyoung mice tolerate treatment of individual mAbs, combination therapywith anti-PD-1 and 10D1 causes severe irAE with multiple organinflammation, anemia and, as shown in FIG. 25 , severely stunted growth.In contrast, when combined with anti-PD-1, chimeric L3D10 exhibits onlymild irAE as demonstrated by normal weight gain.

To further examine to relative toxicity of chimeric L3D10 compared to10D1 when administered in combination with anti-PD-1, we looked at thepathalogical effects in the CTLA4^(h/h) knockin mice at 42 days afteradministration. As shown in FIG. 26 , terminal body weight (day 42) inmice treated with L3D10+anti-PD-1 was similar to mice treated with hIgGnegative control antibody. However, by comparison, the weight of micetreated with 10D1+anti-PD-1 was much lower. Accordingly, when we lookedat the gross anatomy of these mice, the Uterus/Ovary/Bladder and thymuswere noticably smaller in mice treated with 10D1+PD-1 (FIG. 27 ). Again,the organs in mice treated with L3D10+anti-PD-1 was comparable to hIgGcontrol. In contrast, the hearts dissected from mice treated with 10D1appeared slightly larger in size with a noticeably whiter appearance. Asa result we decided to look at erythropoiesis within the mice andobserved clear differences in the mice treated with 10D1+anti-PD-1relative to the groups treated with L3D10+anti-PD-1 or control antibody,which were fairly similar. As shown in FIG. 27A, the bone marrow frommice treated with 10D1+anti-PD-1 had a noticeably whiter color and theisolated blood was almost completely white in color (FIG. 28 b ). Inaccordance with this, when we took at closer look at the cellsundergoing the different stages of blood development using CD71 andCD119 markers. Representative FACS profiles are shown in FIG. 28C, whilesummary data are presented in FIG. 28D. These data revealed astatistically significant reduction in the number of cells undergoingStage IV development in the 10D1+anti-PD-1 treated mice (FIG. 28D).

To explore the potential mechanism of anemia in the 10D1-treated mice,we tested if 10D1+PD-1 treatment induces anti-red blood cell antibodies.As shown in FIG. 29 , no anti-red blood cell antibodies are detected.Thus, development of red cell-specific autoantibodies are notresponsible for anemia in the anti-PD-1+10D1-treated mice.

To further determine the toxicology of L3D10 vs 10D1 in combination withanti-PD-1, we performed histological analysis of the heart (FIG. 30 ),lung (FIG. 31 ), salivary gland (FIG. 32 ) and the kidney and liver(FIG. 33 ) following fixation in 10% formalin for at least 24 hours. Ineach of the tissues studied, mice treated with 10D1+anti-PD-1 displayeda high level of T cell infiltration. The toxicity score, based onseverity of inflammation, are summarized in FIG. 34 , which shows thehigh toxicity scores of mice treated with 10D1+anti-PD-1 relative toL3D10+anti-PD-1 which has scores only marginally higher than the hIgGcontrol mouse group.

Example 10: L3D10 has Reduced Binding for Soluble CTLA4

L3D10 and 10D1 display similar binding patterns for plate immobilizedCTLA4 (FIG. 36 ). As a possible explanation for the reduced toxicity ofL3D10 relative to 10D1, particularly the increased T cellinfiltration/activity associated with 10D1, we decided to look at thebinding to soluble CTLA4. We chose to look at this because theassociation between CTLA4 polymorphism and multiple autoimmune diseasesrelates to the defective production of soluble CTLA4 (nature 2003, 423:506-511) and genetic silencing of the sCTLA4 isoform increased the onsetof type I diabetes in mice (Diabetes 2011, 60:1955-1963). Furthermore,soluble CTLA4 (abatacept and belatacept) is a widely used drug forimmune suppression. In accordance with this idea, when we looked at therelative binding to soluble CTLA4, we observed a marked decrease in thebinding of L3D10 (FIG. 37 ).

We have demonstrate that anti-CTLA-4 mAb induce robust tumor injectionin heterozygous Ctla4^(h/m) mice in which only 50% of CTLA-4 moleculescan bind to anti-human CTLA-4 mAbs. To determine if engagement of 50% ofCTLA-4 is sufficient to induce irAE, we treated the Ctla4^(h/m) micewith anti-PD-1+10D1. As shown in FIG. 35 , anti-PD-1+10D1 failed toinduce weight loss in the Ctla4^(h/m) mice. Therefore, irAE and cancerimmunity can be uncoupled genetically.

In vivo activity demonstrates that the L3D10 antibody retains itsanti-tumor activity but displays reduced autoimmune adverse effectobserved with other immunotherapeutic antibodies such as 10D1,indicating it is possible to enhance anti-tumor activity withoutexacerbating autoimmune adverse events. Accordingly, autoimmune sideeffects are not a necessary price for cancer immunity and that it ispossible to uncouple these two activities. Characterization of L3D10demonstrated that its ability to block the interaction of CTLA4 withB7.1 and B7.2 is more effective than by 10D1 and that this relates to adifference in the CTLA4 binding site between the antibodies.Furthermore, L3D10 was fused to a modified human IgG1 Fc domain that hasmutations conferring strong ADCC activity that enhances the therapeuticeffect of the antibody. Further characterization demonstrates that L3D10and 10D1 bind to immobilized CTLA4 with a similar binding profile.However, L3D10 demonstrates much lower binding affinity to soluble CTLA4than 10D1. Taken together, our data demonstrate that antibody L3D10 hasgreat potential for clinical use in treating cancer patients with lesssevere adverse events.

Example 11. Humanization of L3D10

The humanization process begins by generating a homology modeledantibody 3D structure and creating a profile of the parental antibodybased on structure modeling. Acceptor frameworks to utilize wereidentified based on the overall sequence identity across the framework,matching interface position, similarly classed CDR canonical positions,and presence of N-glycosylation sites that would have to be removed. Onelight chain (LC) and one heavy chain (HC) framework were selected forthe humanization design.

Humanized antibodies were designed by creating multiple hybrid sequencesthat fuse select parts of the parental antibody sequence with the humanframework sequences, including grafting of the CDR sequences into theacceptor frameworks. The predicted CDR sequences of the of parentantibody L3D10 are provided as SEQ ID NOS: 21-26 as indicated in Table1A below:

TABLE 1A The predicted CDR sequences of the parental antibody L3D10Antibody Chain CDR SEQ ID NO Variable Light 1 21 2 22 3 23 VariableHeavy 1 24 2 25 3 26

Using the 3D model, these humanized sequences were methodically analyzedby eye and computer modeling to isolate the sequences that would mostlikely retain antigen binding. The goal was to maximize the amount ofhuman sequence in the final humanized antibodies while retaining theoriginal antibody specificity.

Three humanized light chains (LC1, LC2 and LC3) and three humanizedheavy chains (HC1, HC2 and HC3) were designed based on the selectedacceptor frameworks. Each of the three HC or three LC sequences werefrom the same germline, with different back mutations to the murineparental sequence as shown in FIG. 38 . The humanized variable regionamino acid sequences and their optimized coding nucleotide sequence arelisted in Seq ID NOS: 9-20. The CDR2 sequences of both the humanizedheavy and light chains contain amino acid changes relative to theparental L3D10 antibody sequence and are listed in SEQ ID NOS 33-38 asindicated in Table 1B below.

TABLE 1B CDR2 sequences of the humanized antibody variable regions.Antibody Sequence CDR2 Sequence SEQ ID NO HC1 YIWYDGNTNFHPSLKSR 33 HC2YIWYDGNTNFHSSLKSR 34 HC3 YIWYDGNTNFHSPLKSR 35 LC1 AATNLQS 36 LC2 AATNLQD37 LC3 AATSLQS 38

The light and heavy humanized chains can now be combined to createvariant fully humanized antibodies. All possible combinations ofhumanized light and heavy chains were tested for their expression leveland antigen binding affinity to identify antibodies that perform similarto the parental antibody.

A new tool to calculate humanness scores for monoclonal antibodies (24)were used. This score represents how human-like an antibody variableregion sequence looks, which is an important factor when humanizingantibodies. The humanness scores for the parental and humanizedantibodies are shown in Tables 2 and 3 below. Based on our method, forheavy chains a score of 79 or above is indicative of looking human-like;for light chains a score of 86 or above is indicative of lookinghuman-like.

TABLE 2 Humanized light chain information and humanness scores.Full-length (Framework + Framework CDR) Only Chain Name Note Cutoff = 86Cutoff = 90 L2872 Light chain 71.3 78.2 (Chimeric Parental) L3106 (LC1)Regular humanized 86.5 96.8 L3107 (LC2) Regular humanized 83.6 94.0L3108 (LC3) Regular humanized 88.8 98.1

TABLE 3 Humanized heavy chain information and humanness scores.Full-length (Framework + Framework CDR) Only Chain Name Note Cutoff = 79Cutoff = 84 H2872 Parental 62.0 70.3 (Chimeric Parental) H3106 (HC1)Regular humanized 80.4 90.7 H3107 (HC2) Regular humanized 78.9 89.4H3108 (HC3) Regular humanized 80.5 93.0

Full-length antibody genes were constructed by first synthesizing thevariable region sequences. The sequences were optimized for expressionin mammalian cells. These variable region sequences were then clonedinto expression vectors that already contain human Fc domains; for theheavy chain, the hIgG1 (M252Y, S254T, T256E, S298A, E333A, K334A)backbone was utilized. In addition, for comparison the variable regionof the chimeric parental heavy and light chains were constructed asfull-length chimeric chains using the same backbone Fc sequences.

All 9 humanized antibodies underwent 0.01 liter small scale production.The chimeric parental antibody was also scaled-up for direct comparison.Plasmids for the indicated heavy and light chains were transfected intosuspension HEK293 cells using chemically defined media in the absence ofserum to make the antibodies. Whole antibodies in the conditioned mediawere purified using MabSelect SuRe Protein A medium (GE Healthcare). The10 antibodies tested are shown in Table 4 below.

TABLE 4 Ten antibodies produced transiently in HEK293 cells Heavy LightYield Antibody name Chain Chain PP # (mg/L) Humanized HC1 + LC1 H3106L3106 4630 54 Humanized HC1 + LC2 H3106 L3107 4631 50 Humanized HC1 +LC3 H3106 L3108 4632 45 Humanized HC2 + LC1 H3107 L3106 4633 37Humanized HC2 + LC2 H3107 L3107 4634 44 Humanized HC2 + LC3 H3107 L31084635 40 Humanized HC3 + LC1 H3108 L3106 4636 46 Humanized HC3 + LC2H3108 L3107 4637 55 Humanized HC3 + LC3 H3108 L3108 4638 53 ChimericParental H2872 L2872 4629 28

The affinity of 9 humanized antibody combinations and the chimericparental antibody to the antigen (huCTLA4) was evaluated by Octet.Multi-concentration kinetic experiments were performed on the OctetRed96 system (ForteBio). Anti-hIgG Fc biosensors (ForteBio, #18-5064)were hydrated in sample diluent (0.1% BSA in PBS and 0.02% Tween 20) andpreconditioned in pH 1.7 Glycine. The antigen was diluted using a7-point, 2-fold serial dilution starting at 600 nM with sample diluent.All antibodies were diluted to 10 μg/mL with sample diluent and thenimmobilized onto anti-hIgG Fc biosensors for 120 seconds. Afterbaselines were established for 60 seconds in sample diluent, thebiosensors were moved to wells containing the antigen at a series ofconcentrations to measure the association. Association was observed for120 seconds and dissociation was observed for 180 seconds for eachprotein of interest in the sample diluent. The binding affinities werecharacterized by fitting the kinetic sensorgrams to a monovalent bindingmodel (1:1 binding). The full kinetic measurements are summarized inTable 5 below.

TABLE 5 Kinetic measurements of the humanized antibodies and theparental antibody Loading Sample Sample ID ID KD (M) kon(1/Ms) kdis(1/s)Full X{circumflex over ( )}2 Full R{circumflex over ( )}2 PP4629 huCTLA42.3E−09 3.5E+05 8.0E−04 0.0033 0.9981 PP4630 huCTLA4 1.3E−08 1.3E+051.8E−03 0.0127 0.9848 PP4631 huCTLA4 6.9E−09 2.4E+05 1.6E−03 0.01200.9918 PP4632 huCTLA4 1.2E−08 1.6E+05 1.9E−03 0.0109 0.9915 PP4633huCTLA4 7.1E−09 2.0E+05 1.4E−03 0.0106 0.9933 PP4634 huCTLA4 6.8E−092.8E+05 1.9E−03 0.0116 0.9866 PP4635 huCTLA4 8.4E−09 2.4E+05 2.0E−030.0077 0.9934 PP4636 huCTLA4 8.7E−09 2.5E+05 2.2E−03 0.0111 0.9905PP4637 huCTLA4 6.4E−09 3.2E+05 2.1E−03 0.0173 0.9884 PP4638 huCTLA48.1E−09 2.9E+05 2.3E−03 0.0122 0.9920

Example 12. Anti-Tumor Activity of the Humanized Anti-CTLA4 Antibodies

Based on the relative binding affinity and humanness scores, we chose 3antibodies for further evaluation:

PP4631—high affinity and good expression

PP4637—high affinity and good expression

PP4638—slightly lower affinity but highest humanization score

Material for each of these antibodies was produced by transientproduction in HEK293 cells at the 0.1 liter scale followed by protein Apurification. Binding affinity of the purified antibodies was confirmedby Octet analysis as shown in Table 6 below.

TABLE 6 Kinetic measurements of the humanized antibodies and theparental antibody Loading Sample Replicate Sample ID ID KD (M) kon(1/Ms)kdis(1/s) Full X{circumflex over ( )}2 Full R{circumflex over ( )}2 1PP4631 huCTLA4 7.2E−09 2.3E+05 1.6E−03 0.0274 0.9894 1 PP4637 huCTLA47.1E−09 2.7E+05 1.9E−03 0.0294 0.9899 1 PP4638 huCTLA4 9.4E−09 2.3E+052.1E−03 0.0211 0.9919 2 PP4631 huCTLA4 7.4E−09 2.3E+05 1.7E−03 0.01910.9919 2 PP4637 huCTLA4 8.4E−09 2.6E+05 2.2E−03 0.0248 0.9899 2 PP4638huCTLA4 1.1E−08 2.1E+05 2.2E−03 0.0150 0.9934

We evaluated the anti-tumor activity of these three humanized antibodiescompared to 10D1 and the chimeric L3D10 antibody using the syngeneicMC38 mouse tumor model in human CTLA4-knockin mice described in Example5 above. FIG. 39A shows the treatment schedule of the in vivoexperiment; mice were given a total of 4 doses of antibody every 3 daysstarting on day 7 after inoculation. As shown in FIG. 39B, all humanizedantibodies completely eradicated the tumor and were comparable to 10D1.

In a another experiment we evaluated the anti-tumor activity of thehumanized antibodies PP4631 and PP4637 compared to 10D1 and the chimericL3D10 antibody using the syngeneic MC38 mouse tumor model in theheterozygous Ctla4^(h/m) mice described in Example 5 (FIG. 14 ) at twodifferent doses. As shown in FIG. 40 , whereas all mAbs areindistinguishable when used at 30 mcg/mouse/injection (1.5 mg/kg),PP4637 was more effective at 10 mcg/mouse/injection (0.5 mg/kg), whereasPP4631 and 10D1 showed comparable activity.

The anti-tumor activity of the humanized antibodies compared to 10D1 andthe chimeric L3D10 antibody was also demonstrated using the syngeneicB16-F1 melanoma mouse tumor model in human CTLA4-knockin mice as shownin FIG. 41 . Mice were given a total of 3 doses of antibody every 3 daysstarting on day 2 after inoculation. As shown in FIG. 41 , L3D10 and thehumanized antibodies delayed tumor growth and were comparable to 10D1.

Example 13. Humanized Clones of L3D10 Maintain Superior Safety ProfilesOver 10D1

To test if the superior safety profiles of L3D10 can be maintained afterhumanization, we compared PP4631 and PP4637 with 10D1 for their adverseeffects when used in combination with anti-PD-1. As shown in FIG. 42 ,both PP4631 and PP4637 are less toxic than 10D1 when used in combinationwith anti-PD-1.

Consistent with the defective erythropoiesis described in FIG. 28 , micetreated with 10D1 plus anti-PD-1 are anemic based on complete blood cellcounts (CBC), while those that received anti-PD-1+PP4631 andanti-PD-1+PP4637 have largely normal CBC profiles as shown in FIG. 43 .Moreover, analysis of the T cell profiles in the PBL reveal a robustsystemic activation of both CD4 and CD8 T cells in mice that received10D1+anti-PD-1, but not those that received anti-PD-1+PP4631 oranti-PD-1+PP4637 (FIG. 44 ), further supporting the notion thatL3D10-based anti-CTLA-4 mAbs do not cause systemic T cell activation.

Example 14. Binding Characteristics of the Humanized Anti-CTLA4Antibodies

In order to confirm that the humanized antibodies retained their CTLA4binding characteristics, we looked at binding to immobilized and platebound CTLA4. As shown in FIG. 45 , humanization does not affect bindingto immobilized CTLA4 and all 3 humanized antibodies demonstrated similarbinding to the parental chimeric L3D10 antibody. However, humanizationfurther reduces L3D10 binding to soluble CTLA4 as shown in FIG. 46 .Based on reduced binding to soluble CTLA4, it is anticipated that the 3humanized antibodies will induce equal tumor rejection with even lessautoimmune side effects than L3D10.

We have demonstrated that chimeric L3D10 has a 1000-fold higher blockingactivity than 10D1. This raised an interesting possibility that blockingB7-CTLA-4 interactions may explain its lack of irAE. As shown in FIGS.47 and 48 , neither PP4631 nor PP4637 block B7-CTL-A4 interactions invitro and in vivo. The fact that PP4631 and PP4637 show diminished irAEfurther supported the notion that blocking B7-CTLA-4 interaction is notresponsible for improved safety of L3D10.

Given the proposed role for CTLA-4 in the protection against autoimmunediseases, we proposed reduced binding to soluble CTLA-4 as an underlyingmechanism for improved safety profiles. To test this hypothesis, we usedthe growth weight gain among the female mice that receivedanti-PD-1+anti-CTLA-4 mAbs during the perinatal period as the basicindicator for irAE. As shown in FIG. 42 , severe reduction in weightgain was observed in the mice that received both 10D1 and anti-PD-1,whereas those that received PP4637+anti-PD-1 had the lowest irAE,followed by PP4631 and then L3D10. The strict inverse correlation withreduced binding to sCTLA-4 are consistent with the central hypothesis.

Example 15. Processability Evaluation of the Humanized Anti-CTLA4Antibodies

In order to evaluate the development and manufacturing potential of thethree different humanized antibodies, a number of analytical methodswere performed to characterize the different antibodies.

Characteristic Method Production Transient expression in HEK293 cells,followed by 1-step Protein A purification Purity Size exclusionchromatography (SEC) Purity Capillary Electrophoresis (reduced andnon-reduced) Non-Glyco Capillary Electrophoresis (reduced) DeamidationCapillary isoelectric focusing (cIEF) and liquid chromatography-massspectrometry (LC-MS) following DM stress treatment ThermostabilityDifferential Scanning Calorimetry (DSC) Oxidation Peptide mappingBindinq specificity CHO, 293 blank cell FACS

As an initial assessment, the predicted molecular weights andisoelectric point of the three lead candidate antibodies was calculatedbased on amino acid sequences. As shown in Table 7, all antibodies werefairly similar, although antibody had a slightly lower PI.

TABLE 7 Theoretical parameters of the three humanized antibodies ProteinName Theoretical MW (Da) Theoretical PI PP4631 (49647.8 + 23483.1) × 2 =96614.0 7.9 PP4637 (49644.9 + 23483.1) × 2 = 96611.1 7.65 PP4638(49644.9 + 23311.9) × 2 = 96568.7 7.9

Product Yield Assessment

In order to assess the productivity of the different antibodies, HEK293cells were transiently transfected with vectors expressing the heavy andlight chains of the different antibodies. These cells were then culturedin shake flasks for 6 days using serum-free medium. After 6 days, thesupernatant was collected and the antibodies were purified by one-stepProtein A chromatography. As should in Table 8 below, antibodies PP4631and PP4637 demonstrated similar protein yields whereas antibody PP4638was produced at a much lower relative yield.

TABLE 8 Humanized antibody production yield assessment. AntibodyConcentration (mg/mL) OD 260/280 Yield (mg/L) PP4631 1.280 0.53 126PP4637 4.532 0.53 118 PP4638 0.729 0.57 56

In order to assess the purity of the transiently expressed antibodies,samples were analyzed by reducing and non-reducing SDS-PAGE. As shown inFIG. 50 , samples from all 3 antibodies produced gel bands indicative ofan antibody molecule and that the samples were relatively pure followingProtein A purification.

Size Exclusion Chromatography

To further examine the purity and aggregation of the differentantibodies following transient expression, we performed size exclusionchromatography of the purified proteins. Briefly, 50 μg of filtered(using 0.22 μm filter) sample was used for SE-HPLC separation using aTOSOH G3000 SWxl 5 μm column. PBS pH 7.4 was used as the mobile phase.As shown in Table 9 below, all the humanized antibodies show >90% purityafter protein A purification. Antibodies PP4631 and PP4637 demonstratedsimilarly low levels of higher molecular weight (MW) aggregates anddegradation present with the antibody samples with most of the proteinwithin the main peak. In contrast, antibody PP4638 had higher levels ofaggregation and some degradation. The SE-HPLC chromatograms are shown inFIG. 51 .

TABLE 9 Size Exclusion Chromatography Antibody Aggregation Main PeakDegradation PP4631 2.6% 97.4% 0 PP4637 3.0% 97.0% 0 PP4638 6.5% 92.4%1.1%

Capillary Electrophoresis (CE)

Capillary electrophoresis was used to quantitate the amount of proteinwithin the peak bands under both reduced and non-reduced conditions, aswell as the amount of unglycosylated heavy chain protein. Briefly, 100μg of sample was diluted into CE-SDS sample buffer along withlodoacetamide (non-reduced conditions) or P-mercaptoethanol (reducedconditions), along with 2 μL of a 10 kDa standard protein. Samples werethen treated for 10 min at 70° C. For separation, PA-800, 50 μm I.D.bare-fused silica capillary was used; running length 20.2 cm; separatingvoltage 15 kV; OD220 for detection. As shown in Table 10 below, allthree proteins demonstrated high levels of purity, consistent withSDS-PAGE, and all were highly glycosylated. The CE-SDS chromatograms areshown in FIG. 52 .

TABLE 10 Capillary Electrophoresis Unglycosylated Antibody Non-reduced %Reduced % Heavy Chain PP4631 97.3 99.5 0.3 PP4637 97.2 99.5 0.4 PP463896.9 99.4 0.4

Deamidation: Capillary Isoelectric Focusing (cIEF) and LiquidChromatography-Mass Spectrometry (LC-MS)

The level of protein deamidation under high pH stress was determined bycomparing the antibodies with and without high pH stress treatment overtwo different time periods (5 hrs and 12.5 hrs), followed by cIEF andLC-MS analysis.

The charge isoform profile and isoelectric points of the differentantibodies was determined by capillary isoelectric focusing (cIEF).Briefly, samples underwent buffer exchange into 20 mM Tris pH 8.0 andthen 100 μg of sample protein was mixed with the amphoteric electrolyte,methyl cellulose, along with PI 7.05 and PI 9.77 markers. iCE3™ was usedfor analysis, with a 100 μm I.D. capillary; 1.5 kV plus 3 kV; OD280 fordetection. For deamidation stress treatment, samples were treated with500 mM NaHCO₃ for 5 hr or 12.5 hr, then examined with cIEF and LC-MS.The results of the analysis are shown in Table 11 below and the LC-MSgraphs are shown in FIG. 53 . All three antibodies show a predictedincrease in the amount of deamidated species with stress conditions anda corresponding drop in the main peak. As predicted from the amino acidsequence, the pI of antibody PP4637 is a little lower than for PP4631and PP4638 (Table 7) and the higher observed pI compared to thepredicted pI presumably indicates glycosylation.

TABLE 11 Isoelectric focusing and deamidation DM treatment Main AntibodyPeak PI time DM % peak % Basic % PP4631 8.28 Untreated 17.3 71.9 3.0   5h 27.1 65.4 3.0 12.5 h 40.3 53.2 2.5 PP4637 8.11 Untreated 18.1 79.0 2.9  5 h 31.5 65.7 2.8 12.5 h 44.0 53.8 2.2 PP4638 8.36 Untreated 22.5 69.62.3   5 h 34.4 57.6 2.7 12.5 h 45.9 46.5 2.3

Differential Scanning Calorimetry (DSC) Thermal Analysis

In order to determine the thermal stability and melting temperatures ofthe different antibodies, they were subject to Differential ScanningCalorimetry (DSC) Thermal Analysis. Briefly, 2 mg/mL samples in PBS pH7.4 were subject to temperature ramping from 15° C. to 105° C. at a rateof 1° C./min. Cp changing with temperature was monitored for bothsamples and buffer (as background). Cp vs temperature curves wereobtained with background subtraction, and peaks indicated the Tm of theanalytes. As shown in Table 12 below, all three antibodies demonstrateda similarly high melting temperature. DSC curves for the threeantibodies are shown in FIG. 54 .

TABLE 12 Size Exclusion Chromatography Antibody T_(M) (° C.) PP4631 75.6PP4637 76.2 PP4638 76.6

Oxidation: Peptide Mapping

Oxidative modification of the humanized antibodies was evaluated bypeptide mapping using LC-MS with or without oxidative stress. Thesamples were denatured at 65° C. in the presence of 6M GnCl and 5 mMp-ME, then acetylated with iodacetamide. The processed samples are thendigested with Trypsin (Promega, sequencing grade) at 55° C. and thedigested mixture was separated on a C18 reversed phase LC column(ACQUITY UPLC BEH130 C18, 2.1×100 mm, 1.7 μm) and analyzed by massspectrometry (Waters XEVO-G2S QTOF) using Masslynx and Biophatmlynxanalysis tools. For the oxidation stress analysis, samples treated with0.05% or 0.1% H₂O₂ for 1 hr, then examined with LC-MS. The results areshown in Tables 13-16 below.

TABLE 13Oxidation of the humanized antibodies at Methionine sites. Top panel: antibody PP4631.Middle panel: antibody PP4637. Bottom panel: antibody PP4638.PP4631 oxidation (%) Fragment Modification Oxidation 0.05% H₂O₂0.1% H₂O₂ Modifiers Number start end sites Sequence 0 h Oxidation 1 hOxidation 1 h Oxidation M(1) 1:T001   1  18   4DIQMTQSPSSLSASVGDR (SEQ ID NO: 74) 0.4 0.5 0.4 2:T037 425 447 436WQQGNVFSCSVMHEALHNHYTQK (SEQ ID NO: 75 0.6 1.6 2.7 PP4637 oxidation (%)Fragment Modification Oxidation 0.05% H₂O₂ 0.1% H₂O₂ Modifiers Numberstart end sites Sequence 0 h Oxidation 1 h Oxidation 1 h Oxidation M(1)1:T001   1  18   4 DIQMTQSPSSLSASVGDR (SEQ ID NO: 74) 0.4 0.5 0.5 2:T036425 447 436 WQQGNVFSCSVMHEALHNHYTQK (SEQ ID NO: 75) 0.7 1.7 3.0PP4638 oxidation (%) Fragment Modification Oxidation 0.05% H₂O₂0.1% H₂O₂ Modifiers Number start end sites Sequence 0 h Oxidation 1 hOxidation 1 h Oxidation M(1) 1:T001   1  18   4DIQMTQSPSSLSASVGDR (SEQ ID NO: 74) 0.7 0.7 0.6 2:T036 425 447 436WQQGNVFSCSVMHEALHNHYTQK (SEQ ID NO: 75) 0.9 1.6 2.8

TABLE 14Oxidation of the humanized antibody PP4631 at Tryptophan sites. Red numbers indicate evidence offragmentation found; ″—″ indicates none detected; ″0″ indicates detected at extremely low levels.PP4631 oxidation (%) Fragment Modification Oxidation 0.05% H₂O₂0.1% H₂O₂ Modifiers Number start end sites Sequence 0 h Oxidation 1 hOxidation 1 h Oxidation 1:T003  25  39  35ASENIYSNLAWYQQK (SEQ ID NO: 76) 0.2 0.2 0.2 W(1) 1:T007  62 103  92FSGSGSGTDYTLTISSLQPEDFATYFCQHLWGTPYT — 0 0 FGQGTK (SEQ ID NO: 77) 1:T013146 149 148 VQWK (SEQ ID NO: 78) 0.2 0.2 0.1 2:T001   1  38  36QVQLQESGPGLVKPSETLSLTCTVSGFSLTSYGLSWI 0 — — R (SEQ ID NO: 79) 2:T003  44 64  47/52 GLEWIGYIWYDGNTNFHPSLK (SEQ ID NO: 80) 0 0.2 0.2 2:T009  98129 115 TEGHYYGSNYGYYALDYWGQGTSVTVSSASTK 0 0.1 0 (SEQ ID NO: 81) 2:T012156 218 166 DYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLS 0.1 0.1 0.1SVVTVPSSSLGTQTYICNVNHKPSNTK (SEQ ID NO: 82) 2:T020 283 296 285FNWYVDGVEVHNAK (SEQ ID NO: 83) 4.5 3.9 3.9 2:T023 310 325 321VVSVLTVLHQDWLNGK (SEQ ID NO: 84) 0 0 0 2:T033 379 400 389GFYPSDIAVEWESNGQPENNYK (SEQ ID NO: 85) 2.2 2.6 2.7 2:T037 425 447 425WQQGNVFSCSVMHEALHNHYTQK (SEQ ID NO: 75) 0.1 0.1 0.1

TABLE 15Oxidation of the humanized antibody PP4637 at Tryptophan sites. Red numbers indicate evidence offragmentation found; ″—″ indicates none detected; ″0″ indicates detected at extremely low levels.PP4637 oxidation (%) Fragment Modification Oxidation 0.05% H₂O₂0.1% H₂O₂ Modifiers Number start end sites Sequence 0 h Oxidation 1 hOxidation 1 h Oxidation 1:T003  25  39  35ASENIYSNLAWYQQK (SEQ ID NO: 76) 0.2 0.2 0.2 W(1) 1:T007  62 103  92FSGSGSGTDYTLTISSLQPEDFATYFCQHLWGTPY — — 0 TFGQGTK (SEQ ID NO: 77) 1:T013146 149 148 VQWK (SEQ ID NO: 78) 0.1 0.1 0.1 2:T001   1  38  36QVQLQESGPGLVKPSETLSLTCTVSGFSLTSYGLS — 0.1 — WIR (SEQ ID NO: 79) 2:T003 44  64  47/52 GLEWIGYIWYDGNTNFHSPLK (SEQ ID NO: 86) 0 0 0.1 2:T008  98129 115 TEG HYYGSNYGYYALDYWGQGTLVTVSSASTK 0.2 0.2 0.1 (SEQ ID NO: 87)2:T011 156 218 166 DYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSL — 0 0SSVVTVPSSSLGTQTYICNVNHKPSNTK (SEQ ID NO: 82) 2:T019 283 296 285FNWYVDGVEVHNAK (SEQ ID NO: 83) 4.0 4.0 4.0 2:T022 310 325 321VVSVLTVLHQDWLNGK (SEQ ID NO: 84) 0 0 0 2:T032 379 400 389GFYPSDIAVEWESNGQPENNYK (SEQ ID NO: 85) 2.5 2.5 2.7 2:T036 425 447 425WQQGNVFSCSVMHEALHNHYTQK (SEQ ID NO: 75) 0.2 0 0.1

TABLE 16Oxidation of the humanized antibody PP4638 at Tryptophan sites. Red numbers indicate evidence offragmentation found; ″—″ indicates none detected; ″0″ indicates detected at extremely low levels.PP4638 oxidation (%) Fragment Modification Oxidation 0.05% H₂O₂0.1% H₂O₂ Modifiers Number start end sites Sequence 0 h Oxidation 1 hOxidation 1 h Oxidation 1:T003  25  42  35ASENIYSNLAWYQQKPGK (SEQ ID NO: 88) — — — W(1) 1:T006  62 103  92FSGSGSGTDFTLTISSLQPEDFATYYCQHLWGTPY 0 0 0 TFGGGTK (SEQ ID NO: 89) 1:T012146 149 148 VQWK (SEQ ID NO: 78) 0.1 — — 2:T001   1  38  36QVQLQESGPGLVKPSETLSLTCTVSGFSLTSYGLS 0 — — WIR (SEQ ID NO: 79) 2:T003  44 64  47/52 GLEWIGYIWYDGNTNFHSPLK (SEQ ID NO: 86) 0.1 0.2 0.2 2:T008  98129 115 TEG HYYGSNYGYYALDYWGQGTLVTVSSASTK 0.3 0.3 0.4 (SEQ ID NO: 87)2:T011 156 218 166 DYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSL 0 0 0.2SSVVTVPSSSLGTQTYICNVNHKPSNTK (SEQ ID NO: 82) 2:T019 283 296 285FNWYVDGVEVHNAK (SEQ ID NO: 83) 3.7 4.1 4.1 2:T022 310 325 321VVSVLTVLHQDWLNGK (SEQ ID NO: 84) 0 0 0 2:T032 379 400 389GFYPSDIAVEWESNGQPENNYK (SEQ ID NO: 85) 2.6 2.9 2.7 2:T036 425 447 425WQQGNVFSCSVMHEALHNHYTQK (SEQ ID NO: 75) 0 0 0

Binding Specificity

The binding specificity of the different antibodies was determined byassessing the ability to detect non-specific binding to two differentcell lines that do not express CTLA4 (CHO and HEK293) relative to 10D1at two different concentrations. Briefly, 100 μg/mL or 20 μg/mL samples(or reference mAb) in PBS was incubated with 3×10e⁶ cells/ml (CHO orHEK293). FITC labeled rabbit-anti-human-IgG antibody (Boster, Wuhuan,China) was used for detection and the binding of target mAb to cells wasmeasured by FACS. As shown in Table 17 below, antibodies PP4631 andPP4637 demonstrate very low binding and good specificity, whereasantibody PP4638 displayed non-specific binding activity to the controlcell lines.

TABLE 17 Binding Specificity to CHO and HEK293 cell lines MFI SamplesCHO HEK 293 CELL only 3.50583 4.16546 2^(nd) Ab only 4.00062 4.6808310D1 (100 ug/ml) 3.82459 5.49435 10D1 (20 ug/ml) 3.70334 4.95407 PP4631(100 ug/ml) 10.8065 7.76113 PP4631 (20 ug/ml) 5.03402 5.5862 PP4637 (100ug/ml) 15.0944 10.5987 PP4637 (20 ug/ml) 5.89652 5.78233 PP4638 (100ug/ml) 83.4742 36.8002 PP4638 (20 ug/ml) 15.3381 9.86523

Example 16. Epitope Mapping of the L3D10 and Humanized Antibodies

In order to map the CTLA-4 binding epitope of the L3D10 parent antibodyand the humanized variants, PP4631 and PP4637, we took advantage of thefact that the mouse and human CTLA4 proteins are cross-reactive to B7-1,but not to the anti-CTLA-4 antibodies. Accordingly, we designed a numberof mutants of the human CTLA-4Fc protein in which clusters of aminoacids from the human CTLA-4 protein were replaced with amino acids fromthe murine Ctla-4 protein. As the anti-CTLA-4 antibodies used in thisstudy do not bind to murine Ctla-4, binding of the anti-human CTLA-4antibodies should be abolished when key residues of the antibody bindingepitope are replaced with murine amino acids.

DNA vectors encoding 11 CTLA-4Fc mutant proteins (M1-M11)(SEQ ID NOS:40-50) were constructed based on the wild type human CTLA-4Fc sequenceand proteins were produced by transient transfection in HEK293 at the0.01 mL scale followed by one-step Protein A chromatographypurification.

Binding of the anti-CTLA4 antibodies to CTLA4Fc proteins was performedby ELISA. Plates were coated with CTLA-4Fc proteins at 1 μg/mL andbiotinylated antibodies or B7-1 Fc fusion protein were then used insoluble phase in the binding assay, with the amounts of protein boundmeasured using horse-radish peroxidase (HRP)-conjugated streptavidin.

The anti-human CTLA-4 antibodies do not cross react with murine Ctla-4,which presumably reflects differences in the amino acid sequence betweenhuman and mouse CTLA-4 in the extracellular domain. FIG. 55 shows thealignment of the human, macaque and mouse CTLA-4 extracellular domainsand highlight the sequence conservation between human and macaque, whileshowing the numerous differences between the murine and primatesequences. Due to conservation of the MYPPPY binding motif, mouse andhuman CTLA4 proteins are cross-reactive to B7-1 (72).

In order to map the binding epitope of the anti-human CTLA-4 antibodieswe generated a number of non-overlapping CTLA-4Fc mutant proteins thatincorporate clusters of murine-specific amino acids into the humanCTLA-4 sequence. The amino acids incorporated into each of the 11mutants is shown in FIG. 55 , and the amino acids sequences of the WTand mutant CTLA-4Fc proteins is shown in FIG. 56 . These proteins wereproduced by transient transfection in HEK293 cells and the yield isprovided in Table 18. Many of the mutations appear to affect proteinexpression as indicated by their yields relative to the WT humanCTLA-4Fc protein. P_(GP)-4₂,T1

TABLE 18 WT and mutant CTLA-4Fc proteins produced transiently in HEK293cells. Protein name Yield (mg) CTLA-4Fc WT control 0.72 Mutant 1 1.29Mutant 2 0.03 Mutant 3 0.21 Mutant 4 0.11 Mutant 5 1.89 Mutant 6 0.38Mutant 7 0.25 Mutant 8 1.61 Mutant 9 0.01 Mutant 10 0.04 Mutant 11 1.70

The capacity of chimeric L3D10 and the humanized antibodies PP4631 andPP4637 to bind the immobilized CTLA-4Fc mutant constructs was thendetermined by ELISA in which plates were coated with the CTLA-4 mutantconstructs and biotinylated anti-CTLA-4 antibodies, or B7-1Ig controlprotein, were added and binding measured using HRP-conjugatedstreptavidin. The results of binding assays are shown in Tables 19-22.As expected, all 4 binding proteins demonstrated nice dose-dependentbinding for the WT CTLA-4Fc protein. However, mutations that wereintroduced into the M9 and M10 proteins appear to alter the overallstructure and these mutants failed to bind B7-1Fc. Mutations introducedin M2 and M4 also partially altered CTLA-4 conformation as indicated byreduced binding relative to the WT protein. Consistent with this notion,all 4 of these mutants (M2, M4, M9 and M10) were expressed at much loweryield (Table 18). In contrast, using binding to the WT CTLA-4Fc proteinand binding of the B7-1Fc proteins as references, M11 clearly stands outas a protein that is expressed well, binds B7-1 Fc efficiently butfailed to bind two humanized anti-CTLA-4 antibodies. Its binding tooriginal L3D10 is also reduced by approximately 100-fold (Table 20). Asexpected, the mutations that affect the overall confirmation alsoaffected the binding to the anti-CTLA-4 antibodies.

TABLE 19 Integrity of CTLA4Ig mutants as indicated by their binding toB7-1 Ig fusion protein. Binding to CTLA4Fc proteins was performed byELISA, with the amounts of biotinylated protein bound measured byhorse-radish peroxidase (HRP)-conjugated streptavidin. Values shown arethe OD450 measurements. WT = wild type CTLA-4Fc. M1-M11 are CTLA-4Fcmutant proteins. Protein Conc. WT M1 M2 M3 M4 M5 M6 M7 M8 M9 M10 M11 00.193 0.196 0.202 0.184 0.182 0.182 0.184 0.185 0.18 0.172 0.175 0.174 00.19 0.182 0.177 0.175 0.171 0.171 0.173 0.17 0.168 0.164 0.162 0.163 10ng/ml 0.259 0.328 0.204 0.267 0.199 0.286 0.255 0.218 0.293 0.166 0.1670.22 10 ng/ml 0.257 0.311 0.187 0.249 0.184 0.271 0.244 0.22 0.276 0.1540.159 0.217 100 ng/ml 1.137 1.594 0.316 1.087 0.359 1.513 1.093 0.7851.468 0.164 0.164 0.884 100 ng/ml 1.111 1.553 0.299 1.082 0.34 1.2211.049 0.695 1.375 0.155 0.15 1.045 1 ug/ml 2.813 3.147 1.179 3.147 1.3752.877 3.053 2.703 3.253 0.199 0.171 3.053 1 ug/ml 2.651 3.053 0.9862.864 1.413 3.025 2.983 2.716 2.93 0.218 0.172 3.159

TABLE 20 Epitope mapping of chimeric L3D10 antibody. Binding to CTLA4Fcproteins was performed by ELISA, with the amounts of biotinylatedprotein bound measured by horse-radish peroxidase (HRP)-conjugatedstreptavidin. Values shown are the OD450 measurements. WT = wild typeCTLA-4Fc. M1-M11 are CTLA-4Fc mutant proteins Protein Conc. WT M1 M2 M3M4 M5 M6 M7 M8 M9 M10 M11 0 0.202 0.196 0.2 0.187 0.184 0.189 0.1920.198 0.187 0.179 0.179 0.183 0 0.195 0.187 0.185 0.18 0.176 0.176 0.1760.176 0.17 0.166 0.166 0.167 10 ng/ml 1.433 2.47 0.375 0.62 0.507 1.5391.033 0.714 1.233 0.18 0.18 0.202 10 ng/ml 1.518 2.432 0.317 0.587 0.3561.366 0.976 0.738 1.237 0.171 0.169 0.203 100 ng/ml 3.053 3.253 1.3842.318 2.142 2.841 2.699 2.495 2.909 0.295 0.215 0.635 100 ng/ml 3.0253.239 1.164 2.354 1.409 2.991 2.771 2.483 2.841 0.304 0.216 0.759 1ug/ml 3.373 3.268 2.387 3.184 2.651 3.025 3.092 3.147 3.136 0.916 0.8042.841 1 ug/ml 3.114 2.967 2.619 3.124 2.659 3.034 3.072 2.991 3.0340.916 0.868 2.983

TABLE 21 Epitope mapping of humanized antibody PP4631. Binding toCTLA4Fc proteins was performed by ELISA, with the amounts ofbiotinylated protein bound measured by horse-radish peroxidase(HRP)-conjugated streptavidin. Values shown are the OD450 measurements.WT = wild type CTLA-4Fc. M1-M11 are CTLA-4Fc mutant proteins ProteinConc. WT M1 M2 M3 M4 M5 M6 M7 M8 M9 M10 M11 10 ng/ml 0.312 2.264 0.2070.198 0.194 0.407 0.22 0.194 0.247 0.177 0.181 0.172 10 ng/ml 0.29 2.2970.184 0.178 0.174 0.378 0.202 0.185 0.222 0.154 0.16 0.164 100 ng/ml1.077 2.827 0.203 0.27 0.219 1.371 0.459 0.281 0.725 0.171 0.17 0.172100 ng/ml 0.841 3.061 0.194 0.264 0.208 1.589 0.42 0.277 0.801 0.1540.155 0.159 1 ug/ml 2.51 2.881 0.339 0.882 0.473 2.79 1.992 1.169 2.330.175 0.17 0.178 1 ug/ml 2.471 2.958 0.263 1.121 0.573 2.795 2.016 1.2432.642 0.167 0.169 0.185

TABLE 22 Epitope mapping of humanized antibody PP4637. Binding toCTLA4Fc proteins was performed by ELISA, with the amounts ofbiotinylated protein bound measured by horse-radish peroxidase(HRP)-conjugated streptavidin. Values shown are the OD450 measurements.WT = wild type CTLA-4Fc. M1-M11 are CTLA-4Fc mutant proteins ProteinConc. WT M1 M2 M3 M4 M5 M6 M7 M8 M9 M10 M11 10 ng/ml 0.597 2.307 0.1950.544 0.189 1.239 0.603 0.19 0.5 0.373 0.169 0.157 10 ng/ml 0.535 2.2440.162 0.195 0.435 1.188 0.516 0.535 0.47 0.148 0.15 0.152 100 ng/ml1.947 2.632 0.182 0.389 0.248 2.601 1.296 0.521 2.001 0.15 0.15 0.152100 ng/ml 2.229 2.186 0.175 0.364 0.221 2.425 0.875 0.405 2 0.137 0.1390.148 1 ug/ml 2.724 2.05 0.259 1.662 0.725 2.654 2.355 1.418 2.548 0.1570.151 0.162 1 ug/ml 2.742 2.297 0.274 1.549 0.724 2.84 2.374 1.369 2.690.147 0.143 0.165

TABLE 23 Raw data from a repeat study showing specific loss of antigenicepitope only in M11. As in Table 2-5, except that additional controlswere included to shown specificity of the binding. May 3, 2016 Ab ConchCTLA4-Fc M1 M2 M3 M4 M5 M6 M7 M8 M9 M10 M11 Biotin-L3D10 0 0.18 0.1870.377 0.183 0.22 0.177 0.183 0.186 0.368 0.15 0.215 0.171 0 0.177 0.2220.538 0.167 0.18 0.229 0.177 0.142 0.217 0.293 0.114 0.155 10 ng/ml1.705 2.692 0.469 0.623 0.817 1.853 1.244 0.837 1.27 0.158 0.169 0.19 10ng/ml 1.799 2.779 0.333 0.593 0.563 1.802 1.331 0.884 1.454 0.213 0.1570.194 100 ng/ml 3.316 3.195 1.313 2.244 2.233 3.251 3.032 2.672 3.0150.419 0.26 0.752 100 ng/ml 3.458 3.567 1.37 2.535 2.356 3.316 3.0322.875 3.157 0.346 0.272 0.746 1000 ng/ml 3.567 3.509 2.833 3.333 3.083.413 3.282 3.299 3.352 1.124 0.945 2.888 1000 ng/ml 3.672 3.509 2.7553.299 3.145 3.537 3.316 3.352 3.435 1.181 0.941 2.914 Biotin-HB7-1 00.195 0.2 0.202 0.193 0.192 0.197 0.195 0.198 0.192 0.186 0.185 0.186 00.192 0.185 0.181 0.192 0.178 0.178 0.178 0.187 0.173 0.169 0.168 0.16110 ng/ml 0.316 0.37 0.216 0.304 0.22 0.345 0.279 0.258 0.326 0.177 0.1760.239 10 ng/ml 0.31 0.356 0.21 0.414 0.26 0.331 0.279 0.253 0.297 0.1590.167 0.236 100 ng/ml 1.581 1.882 0.333 1.245 0.527 1.813 1.235 0.8991.557 0.176 0.172 1.092 100 ng/ml 1.525 1.928 0.323 1.345 0.489 1.7351.385 0.987 1.643 0.162 0.155 1.283 1000 ng/ml 3.76 3.6 1.167 3.4351.973 3.316 3.413 3.101 3.635 0.232 0.185 3.568 1000 ng/ml 3.6 3.6731.316 3.51 2.009 3.459 3.413 3.183 3.635 0.215 0.181 3.673 Biotin-HL1210 ng/ml 0.451 2.812 0.207 0.202 0.194 0.626 0.23 0.207 0.327 0.1970.205 0.181 10 ng/ml 0.417 2.693 0.181 0.179 0.177 0.642 0.22 0.195 0.320.158 0.182 0.162 100 ng/ml 1.868 3.568 0.212 0.29 0.256 2.618 0.5890.345 1.532 0.172 0.174 0.171 100 ng/ml 1.938 3.317 0.203 0.274 0.2472.126 0.571 0.305 1.419 0.155 0.155 0.162 1000 ng/ml 2.99 3.568 0.2681.181 0.712 2.922 2.187 1.329 2.817 0.181 0.17 0.177 1000 ng/ml 3.0333.51 0.268 1.184 0.759 3.071 2.358 1.475 2.869 0.144 0.171 0.187Biotin-HL32 10 ng/ml 0.983 2.654 0.202 0.218 0.197 1.409 0.429 0.2180.727 0.176 0.176 0.17 10 ng/ml 0.955 2.604 0.184 0.2 0.168 1.359 0.3890.21 0.761 0.148 0.154 0.152 100 ng/ml 2.669 3.007 0.232 0.534 0.3192.908 1.839 0.523 2.669 0.145 0.161 0.16 100 ng/ml 2.741 3.158 0.2030.554 0.374 2.895 1.741 0.478 2.604 0.145 0.148 0.157 1000 ng/ml 3.1833.146 0.327 1.837 1.019 2.966 2.817 1.72 3.042 0.173 0.163 0.174 1000ng/ml 3.209 3.316 0.321 1.867 1.015 3.196 2.857 1.766 3.051 0.143 0.1630.187 Biotin-L3D10 Biotin-L3D10 Biotin-HB7-1 Biotin-hB7-1 Ab concmCTLA4-Fc hlg-Fc mCTLA4-Fc hlg-Fc Biotin-HL12 Biotin-HL12 Biotin-HL32 00.19 0.198 0.202 0.191 0 0.189 0.184 0.18 0.185 10 ng/ml 0.201 0.2010.338 0.181 0.179 0.188 0.185 0.179 10 ng/ml 0.18 0.182 0.318 0.1640.165 0.162 0.17 0.181 100 ng/ml 0.303 0.315 1.635 0.176 0.171 0.1770.185 0.176 100 ng/ml 0.314 0.326 1.668 0.165 0.162 0.163 0.165 0.1711000 ng/ml 0.942 1.385 3.569 0.18 0.177 0.182 0.184 0.183 1000 ng/ml0.94 1.475 3.353 0.179 0.172 0.177 0.176 0.187 mCTLA4 hlgG mCTLA4 hlgGmCTLA4 hlgG mCTLA4 hlgG Biotin-L3D10 Biotin-hB7-1 Biotin-HL12Biotin-HL32

Since L3D10 retained significant binding to M11, we tested if thebinding is specific. We coated plate with human CTLA4-Fc (hCTLA4Fc),mouse CTLA4-Fc (mCTLA4-Fc), Control IgG1-Fc or all mutant hCTLA4-Fc andmeasured their binding to B7-1Fc along with L3D10, PP4631 and PP4637.The bulk of the data are presented in Table 23. As shown in FIG. 57 ,biotinylated B7-1 binds hCTLA-4, mCTLA-4 and M11, equally well. Thespecificity of the assay is demonstrated by lack of binding to IgG1-Fc.Interesting, while L3D10-binding to M11 is stronger than those toIgG1-Fc and mCTLA4-Fc, significant binding to IgG1-Fc suggest that thechimeric antibody binding to M11 maybe non-specific. In contrast, noneof the humanized antibodies bind to M11, mCTLA-4, and IgG1-Fc control.These data demonstrate that mutations introduced in M11 selectivelyablated L3D10, PP4631 and PP4637 binding to CTLA-4.

Using known complex structure 133, we mapped the CTLA-4 epitope in a 3-Dstructure. As shown in FIG. 58 , the epitope recognized by these mAbslocalized within the area covered by B7-1. As such, L3D10, PP4631 andPP4637 binding to CTLA-4 would be mutually exclusive to that of B7-1.The poor blocking PP4631 and PP4637 is due to lower avidity rather thandistinctive binding domains.

Taking advantage of the fact that the mouse and human CTLA4 proteins arecross-reactive to B7-1, but that anti-human CTLA-4 antibodies do notcross react with murine Ctla-4 protein, we were able to map the bindingepitope of the L3D10 derived antibodies by ELISA. Using a number ofmutants of the human CTLA-4Fc protein in which clusters of amino acidsfrom the human CTLA-4 protein were replaced with amino acids from themurine Ctla-4 protein, we clearly demonstrate that when we replace 4amino acids that immediately follow the known B7-1 binding domain ofCTLA-4, dose-dependent binding of the antibodies is largely abolished.The fact that the binding epitope maps directly adjacent to the B7-1binding domain correlates well with the demonstrated ability of theL3D10 antibodies to block B7-CTLA-4 interactions both in vitro and invivo. Since soluble CTLA4 is produced by fusion of C-terminal aminoacids of the extracellular IgV domain to intracellular domain, it istempting to speculate that antibody that binds to polymorphic C-terminaldomain residues (only 18 amino acid from the C-terminus) is more likelyto lose reactivity to soluble CTLA-4, in which a large intracellulardomain is fused to the C-terminus of the extracellular domain.

To further investigate the binding domain of the anti-CTLA4 antibodies,6 additional mutant CTLA4-Fc fusion proteins, designated M12-M17 (SEQ IDNOS: 51-56), were designed (FIG. 59 ) and used to compare the binding ofanti-CTLA4 antibodies 10D1 (FIG. 60A), PP4631 (FIG. 60B) and PP4637(FIG. 60C). As shown in FIG. 60 , mutations in M11, which are atpositions Y¹⁰³L¹⁰⁴I¹⁰⁶, abrogated binding to 10D1, PP4631 and PP4637,demonstrating that binding sites for 10D1, PP4631 and PP4637 includeresidues Y¹⁰³L¹⁰⁴I¹⁰⁶. Importantly, additional mutation in A29>Yrestored binding of CTLA-4 with mutations in Y¹⁰³L¹⁰⁴I¹⁰⁶ to PP4631 andPP4637. These data demonstrate that position A²⁹ in CTLA4 is importantfor the binding of antibodies PP4631 and PP4637, but not 10D1.

Example 17. Anti-CTLA-4 mAb Synergizes with Anti-4-1BB in Inducing TumorRejection

Studies in animal models have indicated that the anti-tumor responseelicited by anti-CTLA-4 monoclonal antibody (mAb) is, at least in part,due to an antigen-specific T cell response against normal “self”differentiation antigens (73, 74). The tendency of anti-CTLA-4antibodies to exacerbate autoimmune diseases is well documented in themouse (75-78). This notion was further corroborated and proved to be amajor limitation in more recent human trials in which the patientsdeveloped severe autoimmune manifestations that required discontinuationof treatment (79). On the other hand, cancer therapeutic anti-4-1BB mAbshave been shown to abrogate the development of autoimmune diseases inlupus prone mice (24, 25).

The fact that anti-4-1BB mAbs can both stimulate anti-tumor responsesand decrease autoimmune manifestations raises the intriguing possibilitythat the combination of this antibody with anti-CTLA-4 mAb may result incancer rejection without autoimmunity. In this study anti-CTLA-4 andanti-4-1BB were combined to induce rejection of large establishedtumors.

Combined Effect of Anti-Murine-CTLA-4 and Anti-Murine-4-1BB Antibodiesin the Induction of CD8 T Cell-Mediated Tumor Rejection.

Two models, one of minimal disease and one of large established tumors,were used to test the anti-tumor effect of combining anti-murine-4-1BBand anti-murine-CTLA-4 mAb treatments. C57BL/6 mice were challenged witha subcutaneous inoculation of MC38 colon cancer cells, and at differenttimes after tumor cell inoculation, antibodies were injected intotumor-challenged mice and the tumor size and incidence were monitored byphysical examination.

In the minimal disease model, the mice were treated with hamster IgGplus rat IgG, anti-4-1BB plus hamster IgG (anti-4-1BB alone group),anti-CTLA-4 plus rat IgG (anti-CTLA-4 alone group), or anti-4-1BBcombined with anti-CTLA-4 starting at 48 hours after inoculation oftumor cells. The antibodies were administered intraperitoneally (i.p.)on days 2, 9, and 16. Treatment with either anti-4-1BB or anti-CTLA-4mAb alone resulted in a delay in tumor growth with 1 out of 5 mice ineach group rejecting tumors, while 4 out of 5 mice treated with bothanti-CTLA-4 and anti-4-1BB mAbs were tumor-free at the conclusion of theexperiment. FIG. 61A displays the tumor growth measurements for eachmouse. To compare growth rates between groups, a linear random-effectsmodel was applied to the data. The combination therapy significantlyreduced the daily growth in tumor size by 4.6 mm²/day over anti-CTLA-4alone (p=0.0094). Furthermore, the combination therapy significantlyreduced growth by 8.4 mm²/day over anti-4-1BB alone (p=0.0006). Inaddition to the growth rate, the actual tumor sizes were comparedbetween the treatment groups at six weeks after the initial tumorchallenge. The average tumor size at six weeks was significantly smallerfor mice given the combination therapy (27.5 mm²) compared to mice giveneither anti-CTLA-4 (137.8 mm², p=0.0251) or anti-4-1BB separately(287.6, p=0.0006). Thus, in the setting of minimal tumor-burden, thecombination of anti-4-1BB and anti-CTLA-4 mAbs results in significantdelays in tumor growth over anti-4-1BB or anti-CTLA-4 given separately.

To determine if the anti-tumor effects of combination mAb treatmentagainst small tumor burden could be extended to therapeutic applicationsagainst larger tumor burdens, mice with established tumors were treatedwith antibodies. Wild type C57BL/6 mice were challenged with asubcutaneous inoculation of MC38 colon cancer cells. Tumors were allowedto grow for 14 days, at which point, mice with established tumors(usually >7 mm in diameter) were selected and divided randomly into fourtreatment groups: hamster IgG plus rat IgG, anti-4-1BB plus hamster IgG,anti-CTLA-4 plus rat IgG, and anti-4-1BB mAb combined with anti-CTLA-4mAb. The antibodies were administered i.p. on days 14, 21, and 28 aftertumor challenge. As shown in FIG. 61B, treatment with anti-CTLA-4 mAbdid not impede tumor growth when compared to control IgG treatment,although rejection was seen in one of the eight mice in the group.Treatment with anti-4-1BB mAb slowed tumor growth somewhat, but only onein eight mice rejected the tumor. In contrast, combination therapy withboth anti-CTLA-4 and anti-4-1BB mAbs led to the eradication of tumors in7 out of 8 mice and prevention of further tumor growth in the remainingmouse. As above, growth rates between groups were compared by applying alinear random-effects model to the data. The combination therapysignificantly reduced the daily growth in tumor size by 10.6 mm²/dayover anti-CTLA-4 alone (p<0.0001). Furthermore, the combination therapysignificantly reduced growth by 6.2 mm²/day over anti-4-1BB alone(p=0.0002). In addition to the growth rate, the actual tumor sizes werecompared between the treatment groups at eight weeks after the initialtumor challenge. The estimated average tumor size at eight weeks wassignificantly smaller for mice given the combination therapy (−1.7 mm²,95% CI: −10.8, 7.5 mm²) compared to mice given either anti-CTLA-4 (404.9mm², 95% CI: 285.4, 524.4 mm²; p<0.0001) or anti-4-1BB separately (228.4mm², 95% CI: 200.4, 689.9 mm²; p=0.0004). Therefore, the combination mAbalso appears to significantly delay tumor growth over anti-CTLA-4 oranti-4-1BB separately in larger tumor burdens as well.

MC38 is known to form liver metastasis.⁸⁰ To evaluate the effect oftherapeutic antibodies on liver metastasis, all mice enrolled in theexperiments were analyzed for liver metastasis by histology. As shown inTable 24, approximately 60% of the control Ig-treated mice hadmicro-metastasis in the liver. Treatments with either anti-CTLA-4 oranti-4-1BB antibodies alone reduced the rate of metastasis somewhat,although the reduction did not reach statistical significance.Remarkably, only 1/22 mice in the group treated with both antibodies hadliver metastases. Using a logistic regression model, we found that theodds of liver metastasis for mice given anti-4-1BB alone wereapproximately 4.7 times higher than the odds for mice given bothanti-4-1BB and anti-CTLA-4 (95% CI: 1.6, 13.7; p=0.0050). Similarly, theodds of liver metastasis were 3.6 times higher for mice givenanti-CTLA-4 only compared to mice given both treatments (95% CI: 1.3,10.2; p=0.0174). Thus, combination therapy significantly reduces livermetastasis by MC38 when compared to treatment with either antibodyalone.

TABLE 24 Combination therapy substantially reduces liver metastases*Number of mice with Group metastasis comparison Group Treatment n (%)p-value G1  Hamster IgG + 19 11 Rat IgG (57.8%) G2 Anti-CTLA-4 + 18  6vs.G1: 0.1383 Rat IgG (33.3%) G3  Anti-4-16B + 21  8 vs.G1: 0.2136Hamster IgG (38.1%) G4 Anti-CTLA-4 + 22  1 vs.G1: 0.0007 Anti-4-1BB(4.5%) vs.G2: 0.0174 vs.G3: 0.0050 *Data are summarized from 4independent experiments. At least two sections per liver were examinedafter H&E staining.

To determine which subset of immune cells was contributing to theanti-tumor effect elicited by combination mAb treatment, the majorsubsets of lymphocytes were depleted with monoclonal antibodies. MC38tumor cells were injected subcutaneously. Once tumors were palpable,tumor-bearing mice were separated into four groups. Each group had aseries of intraperitoneal antibody injections to deplete differingsubsets of immune cells, including no depletion with normal rat IgG, CD4T cell depletion with anti-CD4 mAb (GK 1.5), CD8 T cell depletion withanti-CD8 mAb (2.4.3), and NK cell depletion with anti-NK1.1 mAb (PK136).In addition, all mice in all groups were treated with anti-CTLA-4 plusanti-4-1BB mAbs once weekly for three weeks. Adequate depletion ofimmune cell subsets was evaluated by flow cytometry of peripheral bloodtaken from mice immediately prior to completion of the experiment (datanot shown). As expected, mice with no depletion of immune cellsresponded to treatment with anti-CTLA-4 combined with anti-4-1BB mAb(FIG. 62 ). Similarly, depletion of NK cells and CD4 T cells did notaffect the anti-tumor activity of combination anti-CTLA-4 plusanti-4-1BB mAb therapy. The depletion of CD8 T cells, however, abrogatedthe anti-tumor activity of combination antibody therapy. At day 28, theestimated average tumor size for mice with depletion of CD8 T cells(92.3 mm², 95% CI: 64.5, 120.1 mm²) was significantly higher than theaverage tumor sizes for mice with no depletion of immune cells (28.7mm², 95% CI: −17.1, 74.4 mm²), mice with depleted CD4 T cells (16.7 mm²,95% CI: 1.0, 32.4 mm²), and mice with depleted NK cells (9.3 mm², 95%CI: −8.3, 26.9 mm²). These data demonstrate that the tumor-eradicatingeffect of anti-CTLA-4 and anti-4-1BB mAb treatment is CD8 Tcell-dependent.

Anti-4-1BB Antibody Reduced Antibody Response to Xenogeneic Anti-CTLA-4Antibodies.

One of the obstacles to repeated antibody therapy is the enhancement ofhost antibody responses to the therapeutic antibodies.⁸¹ Since 4-1BB isknown to reduce antibody response to proteins, we evaluated the effectof anti-4-1BB antibodies on host response to anti-CTLA-4 antibodies. Asshown in FIG. 63 , very little, if any anti-antibody response wasdetected in mice treated with either control IgG or anti-4-1BB.Consistent with the ability of anti-CTLA-4 mAb to facilitate CD4 T cellresponses⁸², mice treated with anti-CTLA-4 plus rat IgG developed stronghost antibody responses against the administered 4F10 antibody and ratIgG (FIGS. 63A-B). This response was reduced by more than 30-fold whenanti-4-1BB was co-administered with anti-CTLA-4 mAb. These data suggestthat anti-4-1BB antibodies can potentially increase the duration ofother co-administrated therapeutic proteins by reducing host responsesto the therapeutics.

In Human CTLA-4 Knock-in Mice, a Combination of Anti-Mouse 4-1BB andAnti-Human CTLA-4 Antibodies Induced Tumor Rejection and Long-LastingCancer Immunity.

Since anti-4-1BB reduces the production of antibodies against theanti-CTLA-4 antibodies, an interesting issue is whether the enhancementof tumor rejection by anti-4-1BB is solely due to its effect insuppressing antibody response. This human CTLA4 gene knock-in mouseallowed us to test if the anti-tumor effect of the anti-human CTLA4antibodies can be enhanced by anti-4-1BB antibody. As shown in FIG. 64A,while both anti-human CTLA-4 (L3D10) and anti-4-1BB antibody (2A) alonecaused delayed tumor growth, a combination of the two antibodiesresulted in the most significant tumor rejection. Respectively, in thegroups treated with anti-CTLA-4, 4-1BB or the two antibodies, 1/7, 2/7,5/7 mice never developed tumors, while all mice in the untreated groupdeveloped tumors. Since the anti-human CTLA-4 antibody is of mouseorigin, the impact of 4-1BB antibody cannot be attributed to itssuppression of antibodies to therapeutic anti-CTLA-4 antibodies.Moreover, our data also demonstrated that the superior effect ofcombination therapy will likely be applicable to anti-human CTLA-4antibody-based immunotherapy.

To test whether the double antibody treated mice were immune to furthertumor cell challenge, we challenged them with tumor cells at 110 daysafter their first tumor cell challenge. As shown in FIG. 64B, all of thefive double antibody-treated mice that had rejected the tumor cells inthe first round remained tumor-free, while control naïve mice hadprogressive tumor growth. Thus, combination therapy also inducedlong-lasting immunity to the cancer cells.

One of the obstacles to protein-based immunotherapy is host immunity tothe therapeutic proteins. In the case of antibodies, the host can mountantibodies to xenotypic, allotypic and idiotypic epitopes.⁸¹ Thexenotypic response can be eliminated by complete humanization, althoughother anti-antibody responses require special considerations. Theobstacle is more obvious for anti-CTLA-4 antibody as it is an adjuvantin itself. Previous work by Mittler et al. demonstrated a significantsuppression of T-cell dependent humoral immune response.⁸³ Our datademonstrate that co-administration of anti-4-1BB antibodies reduces hostresponses to the anti-CTLA-4 antibody, which suggests another advantageof combination therapy using anti-CTLA-4 and anti-4-1BB antibody.

Taken together, our data demonstrate that combination therapy withanti-CTLA-4 and anti-4-1BB antibodies offers three major advantages,namely, an increased effect in cancer immunity, mutual suppression ofautoimmune side effects, and amelioration of anti-antibody responses.

All publications and patents mentioned in this specification are hereinincorporated by reference to the same extent as if each individualpublication or patent application was specifically and individuallyindicated to be incorporated by reference in its entirety. While theinvention has been described in connection with specific embodimentsthereof, it will be understood that it is capable of furthermodifications and this application is intended to cover any variations,uses, or adaptations of the invention following, in general, theprinciples of the invention and including such departures from thepresent disclosure as come within known or customary practice within theart to which the invention pertains and as may be applied to theessential features hereinbefore set forth.

REFERENCES CITED

-   1. Townsend ARM, Tothbard J, Gotch F M, Bahadur G, Wraith D,    McMichael A J. The epitope of influenza nucleoprotein recognized by    cytotoxic lymphocytes can be defined with short synthetic peptides.    Cell. 1986; 44:959-68.-   2. Zinkernagel R M, Doherty P C. Restriction of in vitro T    cell-mediated cytotoxicity in lymphocytic choriomeningitis within a    syngeneic or semiallogeneic system. Nature. 1974; 248:701-2.-   3. Lafferty K J, Prowse S J, Simeonovic C J, Warren H S.    Immunobiology of tissue transplantation: a return to the passenger    leukocyte concept. Annu Rev Immunol. 1983; 1:143-73.-   4. Liu Y, Linsley P S. Costimulation of T-cell growth. Curr Opin    Immunol. 1992; 4(3):265-70.-   5. Schwartz R H. Costimulation of T lymphocytes: the role of CD28,    CTLA4, and B7/BB1 in interleukin-2 production and immunotherapy.    Cell. 1992; 71(7):1065-8.-   6. Freeman G J, Freedman A S, Segil J M, Lee G, Whitman J F, Nadler    L M. B7, a new member of the Ig superfamily with unique expression    on activated and neoplastic B cells. J Immunol. 1989;    143(8):2714-22.-   7. Freeman G J, Gribben J G, Boussiotis V A, Ng J W, Restivo V A,    Jr., Lombard L A, et al. Cloning of B7-2: a CTLA4 counter-receptor    that costimulates human T cell proliferation [see comments].    Science. 1993; 262(5135):909-11.-   8. Hathcock K S, Laszlo G, Dickler H B, Bradshaw J, Linsley P, Hodes    R J. Identification of an alternative CTLA4 ligand costimulatory for    T cell activation [see comments]. Science. 1993; 262(5135):905-7.-   9. Wu Y, Guo Y, Liu Y. A major costimulatory molecule on    antigen-presenting cells, CTLA4 ligand A, is distinct from B7. J Exp    Med. 1993; 178(5): 1789-93.-   10. Leach D R, Krummel M F, Allison J P. Enhancement of antitumor    immunity by CTLA4 blockade [see comments]. Science. 1996; 271(5256):    1734-6.-   11. Linsley P S, Brady W, Urnes M, Grosmaire L S, Damle N K,    Ledbetter J A. CTLA4 is a second receptor for the B cell activation    antigen B7. J Exp Med. 1991; 174(3):561-9.-   12. Linsley P S, Clark E A, Ledbetter J A. T-cell antigen CD28    mediates adhesion with B cells by interacting with activation    antigen B7/BB-1. Proc Natl Acad Sci USA. 1990; 87(13):5031-5.-   13. Hodi F S, Mihm M C, Soiffer R J, Haluska F G, Butler M, Seiden M    V, et al. Biologic activity of cytotoxic T lymphocyte-associated    antigen 4 antibody blockade in previously vaccinated metastatic    melanoma and ovarian carcinoma patients. Proc Natl Acad Sci USA.    2003; 100(8):4712-7. PubMed PMID: 12682289.-   14. Hodi F S, O'Day S J, McDermott D F, Weber R W, Sosman J A,    Haanen J B, et al. Improved survival with ipilimumab in patients    with metastatic melanoma. N Engl J Med. 2010; 363(8):711-23. Epub    2010/06/08. doi: 10.1056/NEJMoa1003466. PubMed PMID: 20525992;    PubMed Central PMCID: PMC3549297.-   15. Larkin J, Chiarion-Sileni V, Gonzalez R, Grob J J, Cowey C L,    Lao C D, et al. Combined Nivolumab and Ipilimumab or Monotherapy in    Untreated Melanoma. N Engl J Med. 2015; 373(1):23-34. Epub    2015/06/02. doi: 10.1056/NEJMoa1504030. PubMed PMID: 26027431.-   16. Ribas A, Hodi F S, Callahan M, Konto C, Wolchok J.    Hepatotoxicity with combination of vemurafenib and ipilimumab. N    Engl J Med. 2013; 368(14):1365-6. Epub 2013 Apr. 5. doi:    10.1056/NEJMc1302338. PubMed PMID: 23550685.-   17. Delyon J, Mateus C, Lambert T. Hemophilia A induced by    ipilimumab. N Engl J Med. 2011; 365(18):1747-8. Epub 2011/11/04.    doi: 10.1056/NEJMc1110923. PubMed PMID: 22047582.-   18. Fadel F, El Karoui K, Knebelmann B. Anti-CTLA4 antibody-induced    lupus nephritis. N Engl J Med. 2009; 361(2):211-2. Epub 2009/07/10.    doi: 10.1056/NEJMc0904283. PubMed PMID: 19587352.-   19. Kocak E, Lute K, Chang X, May K F, Jr., Exten K R, Zhang H, et    al. Combination therapy with anti-CTL antigen-4 and anti-4-1BB    antibodies enhances cancer immunity and reduces autoimmunity. Cancer    Res. 2006; 66(14):7276-84. Epub 2006/07/20. doi:    10.1158/0008-5472.CAN-05-2128. PubMed PMID: 16849577.-   20. Lute K D, May K F, Lu P, Zhang H, Kocak E, Mosinger B, et al.    Human CTLA4-knock-in mice unravel the quantitative link between    tumor immunity and autoimmunity induced by anti-CTLA4 antibodies.    Blood. 2005. PubMed PMID: 16037385.-   21. May K F, Roychowdhury S, Bhatt D, Kocak E, Bai X F, Liu J Q, et    al. Anti-human CTLA4 monoclonal antibody promotes T cell expansion    and immunity in a hu-PBL-SCID model: a new method for preclinical    screening of costimulatory monoclonal antibodies. Blood. 2005;    105:1114-20. PubMed PMID: 15486062.-   22. Shields R L, Namenuk A K, Hong K, Meng Y G, Rae J, Briggs J, et    al. High resolution mapping of the binding site on human IgG1 for Fc    gamma RI, Fc gamma RII, Fc gamma RIII, and FcRn and design of IgG1    variants with improved binding to the Fc gamma R. J Biol Chem. 2001;    276(9):6591-604. Epub 2000/11/30. doi: 10.1074/jbc.M009483200.    PubMed PMID: 11096108.-   23. Dall'Acqua W F, Woods R M, Ward E S, Palaszynski S R, Patel N K,    Brewah Y A, et al. Increasing the affinity of a human IgG1 for the    neonatal Fc receptor: biological consequences. J Immunol. 2002;    169(9):5171-80. Epub 2002/10/23. PubMed PMID: 12391234.-   24. Gao S H, Huang K, Tu H, Adler A S. Monoclonal antibody humanness    score and its applications. BMC biotechnology. 2013; 13:55. Epub    2013 Jul. 6. doi: 10.1186/1472-6750-13-55. PubMed PMID: 23826749;    PubMed Central PMCID: PMC3729710.-   25. Sun Y, Chen H M, Subudhi S K, et al. Costimulatory    molecule-targeted antibody therapy of a spontaneous autoimmune    disease. Nat Med 2002.8: 1405-13-   26. Foell J, Strahotin S, O'Neil S P, et al. CD137 costimulatory T    cell receptor engagement reverses acute disease in lupus-prone    NZB×NZW F1 mice. J Clin Invest 2003.111: 1505-18-   27. Melero I, Shuford W W, Newby S A, et al. Monoclonal antibodies    against the 4-1BB T-cell activation molecule eradicate established    tumors. Nat Med 1997.3: 682-5-   28. May K F, Jr., Chen L, Zheng P and Liu Y Anti-4-1BB monoclonal    antibody enhances rejection of large tumor burden by promoting    survival but not clonal expansion of tumor-specific CD8+ T cells.    Cancer Res 2002.62: 3459-65-   29. Ye Z, Hellstrom I, Hayden-Ledbetter M, et al. Gene therapy for    cancer using single-chain Fv fragments specific for 4-1BB. Nat Med    2002.8: 343-8-   30. Walunas, T. L., et al., CTLA4 can function as a negative    regulator of T cell activation. Immunity, 1994. 1(5): p. 405-13.-   31. Krummel, M. F. and J.P. Allison, CD28 and CTLA4 have opposing    effects on the response of T cells to stimulation. J Exp Med, 1995.    182(2): p. 459-65.-   32. Anderson, D. E., et al., Paradoxical inhibition of T-cell    function in response to CTLA4 blockade; heterogeneity within the    human T-cell population. Nat Med, 2000. 6(2): p. 211-4.-   33. Coyle, A. J. et al. (2001) “The Expanding B7 Superfamily:    Increasing Complexity In Costimulatory Signals Regulating T Cell    Function,” Nature Immunol. 2(3):203-209.-   34. Sharpe, A. H. et al. (2002) “The B7-CD28 Superfamily,” Nature    Rev. Immunol. 2:116-126.-   35. Collins, M. et al. (2005) “The B7 Family Of Immune-Regulatory    Ligands,” Genome Biol. 6:223.1-223.7.-   36. Flajnik, M. F. et al. (2012) “Evolution Of The B7 Family:    Co-Evolution Of B7H6 And Nkp30, Identification Of A New B7 Family    Member, B7H7, And Of B7's Historical Relationship With The MHC,”    Immunogenetics epub doi.org/10.1007/s00251-012-0616-2.-   37. Martin-Orozco, N. et al. (2007) “Inhibitory Costimulation And    Anti-Tumor Immunity,” Semin. Cancer Biol. 17(4):288-298.-   38. Flies, D. B. et al. (2007) “The New B7s: Playing a Pivotal Role    in Tumor Immunity,” J. Immunother. 30(3):251-260-   39. Ishida, Y. et al. (1992) “Induced Expression Of PD-1, A Novel    Member Of The Immunoglobulin Gene Superfamily, Upon Programmed Cell    Death,” EMBO J. 11:3887-3895.-   40. Agata, Y. et al. (1996) “Expression Of The PD-1 Antigen On The    Surface Of Stimulated Mouse T And B Lymphocytes,” Int. Immunol.    8(5):765-772.-   41. Yamazaki, T. et al. (2002) “Expression Of Programmed Death 1    Ligands By Murine T Cells And APC,” J. Immunol. 169:5538-5545.-   42. Nishimura, H. et al. (2000) “Facilitation Of Beta Selection And    Modification Of Positive Selection In The Thymus Of PD-1-Deficient    Mice,” J. Exp. Med. 191:891-898.-   43. Martin-Orozco, N. et al. (2007) “Inhibitory Costimulation And    Anti-Tumor Immunity,” Semin. Cancer Biol. 17(4):288-298.-   44. Kabat et al., Sequences of Proteins of Immunological Interest,    5th Ed. Public Health Service, National Institutes of Health,    Bethesda, Md. (1991).-   45 Chothia and Lesk, 1987, J. Mol. Biol. 196:901-917.-   46. Padlan, 1991, Molecular Immunology 28(4/5):489-498.-   47. Studnicka et al., 1994, Protein Engineering 7:805.-   48. Roguska et al., 1994, Proc. Natl. Acad. Sci. USA 91:969-   49. Keler, T. et al. Activity and safety of CTLA4 blockade combined    with vaccines in cynomolgus macaques. J. Immunol. 171, 6251-6259    (2003).-   50. Wing, K. et al. CTLA4 control over Foxp3+ regulatory T cell    function. Science 322, 271-275, doi:10.1126/science. 1160062 (2008).-   51. Schwartz, R. S. The new immunology—the end of immunosuppressive    drug therapy? N. Engl. J. Med. 340, 1754-1756,    doi:10.1056/NEJM199906033402209 (1999).-   52. Simpson, T. R. et al. Fc-dependent depletion of    tumor-infiltrating regulatory T cells co-defines the efficacy of    anti-CTLA4 therapy against melanoma. J. Exp. Med. 210, 1695-1710,    doi: 10.1084/jem.20130579 (2013).-   53. Selby, M. J. et al. Anti-CTLA-4 antibodies of IgG2a isotype    enhance antitumor activity through reduction of intratumoral    regulatory T cells. Cancer immunology research 1, 32-42,    doi:10.1158/2326-6066.CIR-13-0013 (2013).-   54. Maker, A. V., Attia, P. & Rosenberg, S. A. Analysis of the    cellular mechanism of antitumor responses and autoimmunity in    patients treated with CTLA4 blockade. J. Immunol. 175, 7746-7754    (2005).-   55. Korman, A. J., Peggs, K. S. & Allison, J. P. Checkpoint blockade    in cancer immunotherapy. Adv. Immunol. 90, 297-339, doi:    10.1016/S0065-2776(06)90008-X (2006).-   56. Ribas, A. et al. Tremelimumab (CP-675,206), a cytotoxic T    lymphocyte associated antigen 4 blocking monoclonal antibody in    clinical development for patients with cancer. Oncologist 12,    873-883, doi:10.1634/theoncologist. 12-7-873 (2007).-   57. Ribas, A. et al. Phase III randomized clinical trial comparing    tremelimumab with standard-of-care chemotherapy in patients with    advanced melanoma. J. Clin. Oncol. 31, 616-622,    doi:10.1200/JCO.2012.44.6112 (2013).-   58. Lee, K. M. et al. Molecular basis of T cell inactivation by    CTLA4 [In Process Citation]. Science 282, 2263-2266 (1998).-   59. Marengere, L. E. et al. Regulation of T cell receptor signaling    by tyrosine phosphatase SYP association with CTLA4 [published errata    appear in Science 1996 Dec. 6; 274(5293)1597 and 1997 Apr. 4;    276(5309):21]. Science 272, 1170-1173 (1996).-   60. Liu, Y. Is CTLA4 a negative regulator for T-cell activation?    Immunol. Today 18, 569-572 (1997).-   61. Tivol, E. A. et al. Loss of CTLA4 leads to massive    lymphoproliferation and fatal multiorgan tissue destruction,    revealing a critical negative regulatory role of CTLA4. Immunity 3,    541-547 (1995).-   62. Waterhouse, P. et al. Lymphoproliferative disorders with early    lethality in mice deficient in CTLA4 [see comments]. Science 270,    985-988 (1995).-   63. Bachmann, M. F., Kohler, G., Ecabert, B., Mak, T. W. & Kopf, M.    Cutting edge: lymphoproliferative disease in the absence of CTLA4 is    not T cell autonomous. J. Immunol. 163, 1128-1131 (1999).-   64. Bachmann, M. F. et al. Normal pathogen-specific immune responses    mounted by CTLA4-deficient T cells: a paradigm reconsidered. Eur. J.    Immunol. 31, 450-458 (2001).-   65. Nguyen, T. V., Ke, Y., Zhang, E. E. & Feng, G. S. Conditional    deletion of Shp2 tyrosine phosphatase in thymocytes suppresses both    pre-TCR and TCR signals. J. Immunol. 177, 5990-5996 (2006).-   66. Qureshi, O. S. et al. Trans-endocytosis of CD80 and CD86: a    molecular basis for the cell-extrinsic function of CTLA-4. Science    332, 600-603, doi:10.1126/science.1202947 (2011).-   67. Ueda, H. et al. Association of the T-cell regulatory gene CTLA4    with susceptibility to autoimmune disease. Nature 423, 506-511    (2003).-   68. Magistrelli, G. et al. A soluble form of CTLA-4 generated by    alternative splicing is expressed by nonstimulated human T cells.    Eur. J. Immunol. 29, 3596-3602,    doi:10.1002/(SICI)1521-4141(199911)29:11 &#60;    3596::AID-1MMU3596&#62; 3.0.CO;2-Y (1999).-   69. Kremer, J. M. et al. Treatment of rheumatoid arthritis by    selective inhibition of T-cell activation with fusion protein    CTLA4Ig. N. Engl. J. Med. 349, 1907-1915, doi: 10.1056/NEJMoa035075    (2003).-   70. Abrams, J. R. et al. CTLA4Ig-mediated blockade of T-cell    costimulation in patients with psoriasis vulgaris. J. Clin. Invest.    103, 1243-1252, doi:10.1172/JC15857 (1999).-   71. Gerold, K. D. et al. The soluble CTLA-4 splice variant protects    from type 1 diabetes and potentiates regulatory T-cell function.    Diabetes 60, 1955-1963, doi:10.2337/db11-0130 (2011).-   72. Peach R J, Bajorath J, Brady W, Leytze G, Greene J, Naemura J,    et al. Complementarity determining region 1 (CDR1)- and    CDR3-analogous regions in CTLA-4 and CD28 determine the binding to    B7-1. J Exp Med. 1994; 180(6):2049-58.-   73. van Elsas, A., Hurwitz, A. A. & Allison, J. P. Combination    immunotherapy of B16 melanoma using anti-cytotoxic T    lymphocyte-associated antigen 4 (CTLA-4) and granulocyte/macrophage    colony-stimulating factor (GM-CSF)-producing vaccines induces    rejection of subcutaneous and metastatic tumors accompanied by    autoimmune depigmentation. J. Exp. Med. 190, 355-366 (1999).-   74. van Elsas, A. et al. Elucidating the autoimmune and antitumor    effector mechanisms of a treatment based on cytotoxic T lymphocyte    antigen-4 blockade in combination with a B16 melanoma vaccine:    comparison of prophylaxis and therapy. J. Exp. Med. 194, 481-489.    (2001).-   75. Karandikar, N. J., Vanderlugt, C. L., Walunas, T. L.,    Miller, S. D. & Bluestone, J. A. CTLA-4: a negative regulator of    autoimmune disease. J. Exp. Med. 184, 783-788 (1996).-   76. Luhder, F., Chambers, C., Allison, J. P., Benoist, C. &    Mathis, D. Pinpointing when T cell costimulatory receptor CTLA-4    must be engaged to dampen diabetogenic T cells. Proc. Natl. Acad.    Sci. U.S.A 97, 12204-12209 (2000).-   77. Hurwitz, A. A., Sullivan, T. J., Sobel, R. A. & Allison, J. P.    Cytotoxic T lymphocyte antigen-4 (CTLA-4) limits the expansion of    encephalitogenic T cells in experimental autoimmune    encephalomyelitis (EAE)-resistant BALB/c mice. Proc. Natl. Acad.    Sci. U.S.A. 99, 3013-3017 (2002).-   78. Piganelli, J. D., Poulin, M., Martin, T., Allison, J. P. &    Haskins, K. Cytotoxic T lymphocyte antigen 4 (CD152) regulates    self-reactive T cells in BALB/c but not in the autoimmune NOD    mouse. J. Autoimmun. 14, 123-131 (2000).-   79. Phan, G. Q. et al. Cancer regression and autoimmunity induced by    cytotoxic T lymphocyte-associated antigen-4 blockade in patients    with metastatic melanoma. Proc Natl Acad Sci U.S.A. 100, 8372-8377    (2003).-   80. Eisenthal, A. et al. Antitumor effects of recombinant    interleukin-6 expressed in eukaryotic cells. Cancer Immunol.    Immunother. 36, 101-107 (1993).-   81. Schroff, R. W., Foon, K. A., Beatty, S. M., Oldham, R. K. &    Morgan, A. C., Jr. Human anti-murine immunoglobulin responses in    patients receiving monoclonal antibody therapy. Cancer Res. 45,    879-885 (1985).-   82. Kearney, E. R. et al. Antigen-dependent clonal expansion of a    trace population of antigen-specific CD4+ T cells in vivo is    dependent on CD28 costimulation and inhibited by CTLA-4. J. Immunol.    155, 1032-1036 (1995).-   83. Mittler, R. S., Bailey, T. S., Klussman, K., Trailsmith, M. D. &    Hoffmann, M. K. Anti-4-1BB monoclonal antibodies abrogate T    cell-dependent humoral immune responses in vivo through the    induction of helper T cell anergy. J. Exp. Med. 190, 1535-1540    (1999).

The invention claimed is:
 1. A method of treating lung cancer in asubject in need thereof, comprising administering a compositioncomprising an anti-CTLA4 antibody or an antigen binding fragment thereofto the subject, wherein the anti-CTLA4 antibody or the antigen bindingfragment is capable of binding to a human CTLA4 protein and comprises:(a) a light chain variable region comprising: (i) a complementaritydetermining region (CDR) 1 comprising the amino acid sequence set forthin SEQ ID NO: 21; (ii) a CDR2 comprising the amino acid sequence setforth in SEQ ID NO: 36, 37 or 38; and, (iii) a CDR3 comprising the aminoacid sequence set forth in SEQ ID NO: 23; and, (b) a heavy chainvariable region comprising: (i) a CDR1 comprising the amino acidsequence set forth in SEQ ID NO: 24; (ii) a CDR2 comprising the aminoacid sequence set forth in SEQ ID NO: 33, 34 or 35; and, (iii) a CDR3comprising the amino acid sequence set forth in SEQ ID NO:
 26. 2. Themethod of claim 1, wherein: (a) the light chain CDR2 comprises the aminoacid sequence set forth in SEQ ID NO: 37 and the heavy chain CDR2comprises the amino acid sequence set forth in SEQ ID NO: 33; (b) thelight chain CDR2 comprises the amino acid sequence set forth in SEQ IDNO: 37 and the heavy chain CDR2 comprises the amino acid sequence setforth in SEQ ID NO: 35; or, (c) the light chain CDR2 comprises the aminoacid sequence set forth in SEQ ID NO: 38 and the heavy chain CDR2comprises the amino acid sequence set forth in SEQ ID NO:
 35. 3. Themethod of claim 1, wherein: (a) the heavy chain variable regioncomprises the amino acid sequence set forth in SEQ ID NO: 62, 63 or 64;and, (b) the light chain variable region comprises the amino acidsequence set forth in SEQ ID NO: 70, 71 or
 72. 4. The method of claim 3,wherein the heavy chain variable region comprises the amino acidsequence set forth in SEQ ID NO: 62 and the light chain variable regioncomprises the amino acid sequence set forth in SEQ ID NO:
 71. 5. Themethod of claim 3, wherein the heavy chain variable region comprises theamino acid sequence set forth in SEQ ID NO: 64 and the light chainvariable region comprises the amino acid sequence set forth in SEQ IDNO:
 71. 6. The method of claim 3, wherein the heavy chain variableregion comprises the amino acid sequence set forth in SEQ ID NO: 64 andthe light chain variable region comprises the amino acid sequence setforth in SEQ ID NO:
 72. 7. The method of claim 1, wherein the anti-CTLA4antibody or the antigen binding fragment is characterized by reducedbinding to soluble CTLA4.
 8. The method of claim 1, wherein thecomposition comprises the antigen binding fragment.
 9. The method ofclaim 1, wherein the composition is a pharmaceutical compositioncomprising a therapeutically effective amount of the anti-CTLA4 antibodyor the antigen binding fragment, and a physiologically acceptablecarrier or excipient.
 10. The method of claim 1, further comprisingadministering an additional agent selected from the group consisting ofan anti-PD-1 antibody and an anti-4-1BB antibody.
 11. The method ofclaim 10, wherein the anti-PD-1 antibody or anti-4-1BB antibody, and theanti-CTLA4 antibody are combined in a single molecule as a bi-specificantibody.
 12. The method of claim 1, wherein the composition inducesstrong deletion of Treg and local T cell activation in tumormicroenvironment but minimal systemic T cell activation.